Method of identifying compounds that bind galanin receptor (GALR2)

ABSTRACT

This invention provides isolated nucleic acids encoding mammalian galanin receptors, isolated galanin receptor proteins, vectors comprising isolated nucleic acid encoding a mammalian galanin receptor, cells comprising such vectors, antibodies directed to a mammalian galanin receptor, nucleic acid probes useful for detecting nucleic acid encoding a mammalian galanin receptor, antisense oligonucleotides complementary to unique sequences of nucleic acid encoding a mammalian galanin receptor, nonhuman transgenic animals which express DNA encoding a normal or a mutant mammalian galanin receptor, as well as methods of determining binding of compounds to mammalian galanin receptors.

This application is a continuation-in-part of PCT/US97/01301, filed Jan.24, 1997, which is a continuation-in-part in the U.S. of U.S. Ser. No.08/721,837, filed Sep. 27, 1996, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 08/626,685 now U.S. Pat. No.5,972,628, and U.S. Ser. No. 08/626,046, now abandoned, both filed Apr.1, 1996, which are continuations-in-part of U.S. Ser. No. 08/590,494,filed Jan. 24, 1996, now abandoned, the contents of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

Throughout this application, various references are referred to withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofthis application, preceding the sequence listing and the claims.

The neuropeptide galanin and its receptors hold great promise as targetsfor the development of novel therapeutic agents. Galanin is widelydistributed throughout the peripheral and central nervous systems and isassociated with the regulation of processes such as somatosensorytransmission, smooth muscle contractility, hormone release, and feeding(for review, see Bartfai et al., 1993). In the periphery galanin isfound in the adrenal medulla, uterus, gastrointestinal tract, dorsalroot ganglia (DRG), and sympathetic neurons. Galanin released fromsympathetic nerve terminals in the pancreas is a potent regulator ofinsulin release in several species (Ahrén and Lindskog, 1992; Boyle etal., 1994), suggesting a potential role for galanin in the etiology ortreatment of diabetes. High levels of galanin are observed in human andrat anterior pituitary where galanin mRNA levels are potentlyupregulated by estrogen (Vrontakis et al., 1987; Kaplan et al., 1988).The presence of galanin in the hypothalamic-pituitary-adrenal axiscoupled with its potent hormonal effects has led to the suggestion thatgalanin may play an integral role in the hormonal response to stress(Bartfai et al., 1993).

Within the CNS galanin-containing cell bodies are found in thehypothalamus, hippocampus, amygdala, basal forebrain, brainstem nuclei,and spinal cord, with highest concentrations of galanin in thehypothalamus and pituitary (Skofitsch and Jacobowitz, 1985; Bennet etal., 1991; Merchenthaler et al., 1993). The distribution of galaninreceptors in the CNS generally complements that of galanin peptide, withhigh levels of galanin binding observed in the hypothalamus, amygdala,hippocampus, brainstem and dorsal spinal cord (Skofitsch et al., 1986;Merchenthaler et al., 1993; see Bartfai et al., 1993). Accordingly,agents modulating the activity of galanin receptors would have multiplepotential therapeutic applications in the CNS. One of the most importantof these is the regulation of food intake. Galanin injected into theparaventricular nucleus (PVN) of the hypothalamus stimulates feeding insatiated rats (Kyrkouli et al., 1990), an effect which is blocked by thepeptide galanin antagonist M40 (Crawley et al., 1993). In freely feedingrats, PVN injection of galanin preferentially stimulates fat-preferringfeeding (Tempel et al., 1988); importantly, the galanin antagonist M40administered alone decreases overall fat intake (Leibowitz and Kim,1992). These data indicate that specific receptors in the hypothalamusmediate the effects of galanin on feeding behavior, and further suggestthat agents acting at hypothalamic galanin receptors may betherapeutically useful in the treatment of human eating disorders.

Galanin receptors elsewhere in the CNS may also serve as therapeutictargets. In the spinal cord galanin is released from the terminals ofsensory neurons as well as spinal interneurons and appears to play arole in the regulation of pain threshold (Wiesenfeld-Hallin et al.,1992). Intrathecal galanin potentiates the anti-nociceptive effects ofmorphine in rats and produces analgesia when administered alone(Wiesenfeld-Hallin et al., 1993; Post et al., 1988); galanin receptoragonists may therefore be useful as analgesic agents in the spinal cord.Galanin may also play a role in the development of Alzheimer's disease.In the hippocampus galanin inhibits both the release (Fisone et al.,1987) and efficacy (Palazzi et al., 1988) of acetylcholine, causing animpairment of cognitive functions (Sundström et al., 1988). Autopsysamples from humans afflicted with Alzheimer's disease reveal agalaninergic hyperinnervation of the nucleus basalis (Chan-Palay, 1988),suggesting a role for galanin in the impaired cognition characterizingAlzheimer's disease. Together these data suggest that a galaninantagonist may be effective in ameliorating the symptoms of Alzheimer'sdisease (see Crawley, 1993). This hypothesis is supported by the reportthat intraventricular administration of the peptide galanin antagonistM35 improves cognitive performance in rats (Ögren et al., 1992). Humangalanin receptors thus provide targets for therapeutic intervention inmultiple CNS disorders.

High-affinity galanin binding sites have been characterized in brain,spinal cord, pancreatic islets and cell lines, and gastrointestinalsmooth muscle in several mammalian species, and all show similaraffinity for ¹²⁵I-porcine galanin (˜0.5-1 nM). Nevertheless, recent invitro and in vivo pharmacological studies in which fragments andanalogues of galanin were used suggest the existence of multiple galaninreceptor subtypes. For example, a galanin binding site in guinea pigstomach has been reported that exhibits high affinity for porcinegalanin (3-29) (Gu, et al. 1995), which is inactive at CNS galaninreceptors. The chimeric galanin analogue M15 (galantide) acts asantagonist at CNS galanin receptors (Bartfai et al., 1991) but as a fullagonist in gastrointestinal smooth muscle (Gu et al., 1993). Similarly,the galanin-receptor ligand M40 acts as a weak agonist in RINm5Finsulinoma cells and a full antagonist in brain (Bartfai et al, 1993a).The pharmacological profile of galanin receptors in RINm5F cells can befurther distinguished from those in brain by the differential affinitiesof [D-Tyr²]- and [D-Phe²]-galanin analogues (Lagny-Pourmir et al.,1989). The chimeric galanin analogue M35 displaces ¹²⁵I-galanin bindingto RINm5F membranes in a biphasic manner, suggesting the presence ofmultiple galanin receptor subtypes, in this cell line (Gregersen et al.,1993).

Multiple galanin receptor subtypes may also co-exist within the CNS.Galanin receptors in the dorsal hippocampus exhibit high affinity forGal (1-15) but not for Gal (1-29) (Hedlund et al., 1992), suggestingthat endogenous proteolytic processing may release bioactive fragmentsof galanin to act at distinct receptors. The rat pituitary exhibitshigh-affinity binding for 125I-Bolton and Hunter (N-terminus)-labeledgalanin (1-29) but not for [¹²⁵I]Tyr²⁶-porcine galanin (Wynick et al.,1993), suggesting that the pituitary galanin receptor is aC-terminus-preferring subtype. Spinal cord galanin binding sites, whilesimilar to those in brain, show an affinity for the chimeric peptideantagonist M35 intermediate between the brain and smooth muscle (Bartfaiet al., 1991), raising the possibility of further heterogeneity.

A galanin receptor cDNA was recently isolated by expression cloning froma human Bowes melanoma cell line (Habert-Ortoli et al., 1994). Thepharmacological profile exhibited by this receptor is similar to thatobserved in brain and pancreas, and on that basis the receptor has beentermed GALR1. The cloned human GALR1 receptor binds native human,porcine and rat galanin with ˜1 nM affinity (K_(i) vs. ¹²⁵I-galanin) andporcine galanin 1-16 at a slightly lower affinity (˜5 nM). Porcinegalanin 3-29 does not bind to the receptor. The GALR1 receptor appearsto couple to inhibition of adenylate cyclase, with half-maximalinhibition of forskolin-stimulated cAMP production by 1 nM galanin, andmaximal inhibition occurring at about 1 μM.

Recently the rat homologue of GALR1 was cloned from the RIN14Bpancreatic cell line (Burgevin, et al., 1995, Parker et al., 1995; Smithet al., in preparation). The pharmacological data reported to date donot suggest substantial differences between the pharmacologic propertiesof the rat and human GALR1 receptors. Localization studies reveal GALR1mRNA in rat hypothalamus, ventral hippocampus, brainstem, and spinalcord (Gustafson et al., 1996), regions consistent with roles for galaninin feeding, cognition, and pain transmission. However, GALR1 appears tobe distinct from the pituitary and hippocampal receptor subtypesdescribed above.

The indication of multiple galanin receptor subtypes within the brainunderscores the importance of defining galanin receptor heterogeneity atthe molecular level in order to develop specific therapeutic agents forCNS disorders. Pharmacological tools capable of distinguishing galaninreceptor subtypes in tissue preparations are only beginning to appear.Several high-affinity peptide-based galanin antagonists have beendeveloped and are proving useful in probing the functions of galaninreceptors (see Bartfai et al., 1993), but their peptide characterprecludes practical use as therapeutic agents. In light of galanin'smultiple neuroendocrine roles, therapeutic agents targeting a specificdisorder must be selective for the appropriate receptor subtype tominimize side effects.

Accordingly, the cloning of the entire family of galanin receptors foruse in target-based drug design programs has been endeavored. Theidentification of non-peptide agents acting selectively only at specificgalanin receptors will be greatly facilitated by the cloning,expression, and characterization of the galanin receptor family.

The isolation by expression cloning of a novel galanin receptor from arat hypothalamic cDNA library, as well as its pharmacologicalcharacterization in a heterologous expression system is now reported.The data provided demonstrate for the first time the existence of a newgalanin receptor subtype, from now on referred to as the GALR2 subtype,or simply, “GALR2.” The cloning of the human homolog of the rat GALR2receptor is also reported. This discovery provides a novel approach,through the use of heterologous expression systems, to develop subtypeselective, high-affinity non-peptide compounds that could serve astherapeutic agents for eating disorders, diabetes, pain, depression,ischemia, and Alzheimer's disease. The presence of both GALR1 and GALR2in rat hypothalamus suggests that multiple galanin receptors may beinvolved in the regulation of feeding. Pathophysiological disordersproposed to be linked to galanin receptor activation include eatingdisorders, diabetes, pain, depression, ischemia, Alzheimer's disease andreproductive disorders. Accordingly, treatment of such disorders may beeffected by the administration of GALR2 receptor-selective compounds.The localization of GALR2 receptors in other parts of the rat brainsuggests that GALR2 receptors may play a role in cognition, analgesia,sensory processing (olfactory, visual), processing of visceralinformation, motor coordination, modulation of dopaminergic activity,neuroendocrine function, sleep disorders, migraine, and anxiety.

SUMMARY OF THE INVENTION

This invention provides an isolated nucleic acid encoding a mammalianGALR2 galanin receptor. This invention further provides a recombinantnucleic acid encoding a mammalian GALR2 galanin receptor.

This invention further provides an isolated nucleic acid encoding amodified GALR2 receptor, which differs from a GALR2 receptor by havingan amino acid(s) deletion, replacement or addition in the thirdintracellular domain.

This invention also provides a purified GALR2 receptor protein.

This invention further provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding the GALR2 receptor contained in plasmid K985.

This invention further provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding the GALR2 receptor contained in plasmid K1045.

This invention also provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within (a) the nucleic acid sequenceshown in FIG. 1 or (b) the reverse complement of the nucleic acidsequence shown in FIG. 1.

This invention also provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding the GALR2 receptor contained in plasmid BO29.

This invention further provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding the GALR2 receptor contained in plasmid BO39.

This invention provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within (a) the nucleic acid sequenceshown in FIG. 10 or (b) the reverse complement of the nucleic acidsequence shown in FIG. 10.

This invention also provides a nucleic acid probe comprising a nucleicacid molecule of at least 15 nucleotides which is complementary to aunique fragment of the sequence of a nucleic acid molecule encoding aGALR2 receptor.

This invention further provides a nucleic acid probe comprising anucleic acid molecule of at least 15 nucleotides which is complementaryto the antisense sequence of a unique fragment of the sequence of anucleic acid molecule encoding a GALR2 receptor.

This invention provides a transgenic nonhuman mammal comprising ahomologous recombination knockout of the native GALR2 receptor.

This invention also provides a process for identifying a chemicalcompound which specifically binds to a GALR2 receptor which comprisescontacting cells containing DNA encoding and expressing on their cellsurface the GALR2 receptor, wherein such cells do not normally expressthe GALR2 receptor, with the compound under conditions suitable forbinding, and detecting specific binding of the chemical compound to theGALR2 receptor.

This invention further provides a process for identifying a chemicalcompound which specifically binds to a GALR2 receptor which comprisescontacting a membrane fraction from a cell extract of cells containingDNA encoding and expressing on their cell surface the GALR2 receptor,wherein such cells do not normally express the GALR2 receptor, with thecompound under conditions suitable for binding, and detecting specificbinding of the chemical compound to the GALR2 receptor.

This invention additionally provides a process for determining whether achemical compound is a GALR2 receptor agonist which comprises contactingcells transfected with and expressing DNA encoding the GALR2 receptorwith the compound under conditions permitting the activation of theGALR2 receptor, and detecting an increase in GALR2 receptor activity, soas to thereby determine whether the compound is a GALR2 receptoragonist.

This invention also provides a process for determining whether achemical compound is a GALR2 receptor agonist which comprises preparinga cell extract from cells transfected with and expressing DNA encodingthe GALR2 receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with the compound under conditionspermitting the activation of the GALR2 receptor, and detecting anincrease in GALR2 receptor activity, so as to thereby determine whetherthe compound is a GALR2 receptor agonist.

This invention further provides a process for determining whether achemical compound is a GALR2 receptor agonist which comprises preparinga cell extract from cells transfected with and expressing DNA encodingthe GALR2 receptor, isolating a membrane fraction from the cell extract,separately contacting the membrane fraction with both the chemicalcompound and GTPγS, and with only GTPγS, under conditions permitting theactivation of the GALR2 receptor, and detecting GTPγS binding to themembrane fraction, an increase in GTPγS binding in the presence of thecompound indicating that the chemical compound activates the GALR2receptor.

This invention provides a process for determining whether a chemicalcompound is a GALR2 receptor antagonist which comprises contacting cellstransfected with and expressing DNA encoding the GALR2 receptor with thecompound in the presence of a known GALR2 receptor agonist, underconditions permitting the activation of the GALR2 receptor, anddetecting a decrease in GALR2 receptor activity, so as to therebydetermine whether the compound is a GALR2 receptor antagonist.

This invention also provides a process for determining whether achemical compound is a GALR2 receptor antagonist which comprisespreparing a cell extract from cells transfected with and expressing DNAencoding the GALR2 receptor, isolating a membrane fraction from the cellextract, contacting the membrane fraction with the ligand in thepresence of a known GALR2 receptor agonist, under conditions permittingthe activation of the GALR2 receptor, and detecting a decrease in GALR2receptor activity, so as to thereby determine whether the compound is aGALR2 receptor antagonist.

This invention further provides a process involving competitive bindingfor identifying a chemical compound which specifically binds to a GALR2receptor which comprises separately contacting cells expressing on theircell surface the GALR2 receptor, wherein such cells do not normallyexpress the GALR2 receptor, with both the chemical compound and a secondchemical compound known to bind to the receptor, and with only thesecond chemical compound, under conditions suitable for binding of bothcompounds, and detecting specific binding of the chemical compound tothe GALR2 receptor, a decrease in the binding of the second chemicalcompound to the GALR2 receptor in the presence of the chemical compoundindicating that the chemical compound binds to the GALR2 receptor.

This invention provides a process involving competitive binding foridentifying a chemical compound which specifically binds to a humanGALR2 receptor which comprises separately contacting a membrane fractionfrom a cell extract of cells expressing on their cell surface the GALR2receptor, wherein such cells do not normally express the GALR2 receptor,with both the chemical compound and a second chemical compound known tobind to the receptor, and with only the second chemical compound, underconditions suitable for binding of both compounds, and detectingspecific binding of the chemical compound to the GALR2 receptor, adecrease in the binding of the second chemical compound to the GALR2receptor in the presence of the chemical compound indicating that thechemical compound binds to the GALR2 receptor.

This invention also provides a process for determining whether achemical compound specifically binds to and activates a GALR2 receptor,which comprises contacting cells producing a second messenger responseand expressing on their cell surface the GALR2 receptor, wherein suchcells do not normally express the GALR2 receptor, with the chemicalcompound under conditions suitable for activation of the GALR2 receptor,and measuring the second messenger response in the presence and in theabsence of the chemical compound, a change in the second messengerresponse in the presence of the chemical compound indicating that thecompound activates the GALR2 receptor.

This invention further provides a process for determining whether achemical compound specifically binds to and activates a GALR2 receptor,which comprises contacting a membrane fraction isolated from a cellextract of cells producing a second messenger response and expressing ontheir cell surface the GALR2 receptor, wherein such cells do notnormally express the GALR2 receptor, with the chemical compound underconditions suitable for activation of the GALR2 receptor, and measuringthe second messenger response in the presence and in the absence of thechemical compound, a change in the second messenger response in thepresence of the chemical compound indicating that the compound activatesthe GALR2 receptor.

This invention also provides a process for determining whether achemical compound specifically binds to and inhibits activation of aGALR2 receptor, which comprises separately contacting cells producing asecond messenger response and expressing on their cell surface the GALR2receptor, wherein such cells do not normally express the GALR2 receptor,with both the chemical compound and a second chemical compound known toactivate the GALR2 receptor, and with only the second chemical compound,under conditions suitable for activation of the GALR2 receptor, andmeasuring the second messenger response in the presence of only thesecond chemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change in the secondmessenger response in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the GALR2 receptor.

This invention further provides a process for determining whether achemical compound specifically binds to and inhibits activation of aGALR2 receptor, which comprises separately contacting a membranefraction from a cell extract of cells producing a second messengerresponse and expressing on their cell surface the GALR2 receptor,wherein such cells do not normally express the GALR2 receptor, with boththe chemical compound and a second chemical compound known to activatethe GALR2 receptor, and with only the second chemical compound, underconditions suitable for activation of the GALR2 receptor, and measuringthe second messenger response in the presence of only the secondchemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change in the secondmessenger response in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the GALR2 receptor.

This invention also provides a method of screening a plurality ofchemical compounds not known to bind to a GALR2 receptor to identify acompound which specifically binds to the GALR2 receptor, which comprises

(a) contacting cells transfected with and expressing DNA encoding theGALR2 receptor with a compound known to bind specifically to the GALR2receptor;

(b) contacting the preparation of step (a) with the plurality ofcompounds not known to bind specifically to the GALR2 receptor, underconditions permitting binding of compounds known to bind the GALR2receptor;

(c) determining whether the binding of the compound known to bind to theGALR2 receptor is reduced in the presence of the compounds, relative tothe binding of the compound in the absence of the plurality ofcompounds; and if so

(d) separately determining the binding to the GALR2 receptor of eachcompound included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the GALR2 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a GALR2 receptor to identify a compoundwhich specifically binds to the GALR2 receptor, which comprises

(a) preparing a cell extract from cells transfected with and expressingDNA encoding the GALR2 receptor, isolating a membrane fraction from thecell extract, contacting the membrane fraction with a compound known tobind specifically to the GALR2 receptor;

(b) contacting the preparation of step (a) with the plurality ofcompounds not known to bind specifically to the GALR2 receptor, underconditions permitting binding of compounds known to bind the GALR2receptor;

(c) determining whether the binding of the compound known to bind to theGALR2 receptor is reduced in the presence of the compounds, relative tothe binding of the compound in the absence of the plurality ofcompounds; and if so

(d) separately determining the binding to the GALR2 receptor of eachcompound included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the GALR2 receptor.

This invention further provides a method of screening a plurality ofchemical compounds not known to activate a GALR2 receptor to identify acompound which activates the GALR2 receptor which comprises

(a) contacting cells transfected with and expressing the GALR2 receptorwith the plurality of compounds not known to activate the GALR2receptor, under conditions permitting activation of the GALR2 receptor;

(b) determining whether the activity of the GALR2 receptor is increasedin the presence of the compounds; and if so

(c) separately determining whether the activation of the GALR2 receptoris increased by each compound included in the plurality of compounds, soas to thereby identify the compound which activates the GALR2 receptor.

This invention further provides a method of screening a plurality ofchemical compounds not known to activate a GALR2 receptor to identify acompound which activates the GALR2 receptor which comprises

(a) preparing a cell extract from cells transfected with and expressingDNA encoding the GALR2 receptor, isolating a membrane fraction from thecell extract, contacting the membrane fraction with the plurality ofcompounds not known to activate the GALR2 receptor, under conditionspermitting activation of the GALR2 receptor;

(b) determining whether the activity of the GALR2 receptor is increasedin the presence of the compounds; and if so

(c) separately determining whether the activation of the GALR2 receptoris increased by each compound included in the plurality of compounds, soas to thereby identify the compound which activates the GALR2 receptor.

This invention also provides a method of screening a plurality ofchemical compounds not known to inhibit the activation of a GALR2receptor to identify a compound which inhibits the activation of theGALR2 receptor, which comprises

(a) contacting cells transfected with and expressing the GALR2 receptorwith the plurality of compounds in the presence of a known GALR2receptor agonist, under conditions permitting activation of the GALR2receptor;

(b) determining whether the activation of the GALR2 receptor is reducedin the presence of the plurality of compounds, relative to theactivation of the GALR2 receptor in the absence of the plurality ofcompounds; and if so

(c) separately determining the inhibition of activation of the GALR2receptor for each compound included in the plurality of compounds, so asto thereby identify the compound which inhibits the activation of theGALR2 receptor.

This invention further provides a method of screening a plurality ofchemical compounds not known to inhibit the activation of a GALR2receptor to identify a compound which inhibits the activation of theGALR2 receptor, which comprises

(a) preparing a cell extract from cells transfected with and expressingDNA encoding the GALR2 receptor, isolating a membrane fraction from thecell extract, contacting the membrane fraction with the plurality ofcompounds in the presence of a known GALR2 receptor agonist, underconditions permitting activation of the GALR2 receptor;

(b) determining whether the activation of the GALR2 receptor is reducedin the presence of the plurality of compounds, relative to theactivation of the GALR2 receptor in the absence of the plurality ofcompounds; and if so

(c) separately determining the inhibition of activation of the GALR2receptor for each compound included in the plurality of compounds, so asto thereby identify the compound which inhibits the activation of theGALR2 receptor.

This invention also provides a method of modifying feeding behavior of asubject which comprises administering to the subject an amount of acompound which is a GALR2 receptor agonist or antagonist effective toincrease or decrease the consumption of food by the subject so as tothereby modify feeding behavior of the subject.

This invention provides a method for determining whether a compound is aGALR2 antagonist which comprises:

(a) administering to an animal a GALR2 agonist and measuring the amountof food intake in the animal;

(b) administering to a second animal both the GALR2 agonist and thecompound, and measuring the amount of food intake in the second animal;and

(c) determining whether the amount of food intake is reduced in thepresence of the compound relative to the amount of food intake in theabsence of the compound, so as to thereby determine whether the compoundis a GALR2 antagonist.

This invention further provides a method of decreasing feeding behaviorof a subject which comprises administering a compound which is a GALR2receptor antagonist and a compound which is a Y5 receptor antagonist,the amount of such antagonists being effective to decrease the feedingbehavior of the subject.

This invention further provides a method of decreasing nociception in asubject which comprises administering to the subject an amount of acompound which is a GALR2 receptor agonist effective to decreasenociception in the subject.

This invention provides a method of treating pain in a subject whichcomprises administering to the subject an amount of a compound which isa GALR2 receptor agonist effective to treat pain in the subject.

This invention further provides a method of treating Alzheimer's diseasein a subject which comprises administering to the subject an amount of acompound which is a GALR2 receptor antagonist effective to treatAlzheimer's disease in the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Nucleotide coding sequence of the rat hypothalamic galanin GALR2receptor (SEQ ID No: 7), with partial 5′ and 3′ untranslated sequences.Start (ATG) and stop (TAA) codons are underlined.

FIG. 2 Deduced amino acid sequence of the rat hypothalamic galanin GALR2receptor encoded by the nucleotide sequence shown in FIG. 1 (SEQ ID No:8).

FIGS. 3A-3C3A. Diagram of the intron-exon arrangement of the rat GALR2receptor cDNA contained in plasmid K985. Untranslated regions areindicated by dark hatched segments, and coding region is unmarked exceptfor light gray hatched segments indicating the location of thetransmembrane domains of the rat GALR2 receptor. The black segmentindicates the location of the intron. 3B. Splice junction sequences ofthe rat GALR2 receptor. Nucleotide number 1 is located 45 nucleotidesupstream of the start codon (SEQ ID No: 9). 3C. Intron sequence of ratGALR2 receptor cDNA contained in plasmid K985. Nucleotide number 1 islocated 45 nucleotides upstream of the start codon (SEQ ID NO: 9).

FIGS. 4A-4C Localization of [¹²⁵I]galanin binding sites in rat CNS.FIGS. 4A-1 and 4A-4: Distribution of total [¹²⁵I]galanin binding incoronal sections through the hypothalamus and amygdala. FIGS. 4A-2 and4A-5: Binding which remains in these areas following incubation with 60nM [D-Trp²]galanin₍₁₋₂₉₎. FIGS. 4A-3 and 4A-6: Binding obtained afterincubation with 5 μM porcine galanin, which represents the non-specificbinding condition. FIG. 4B: FIGS. 4B-1 to 4B-8: Higher magnificationphotomicrographs of the [¹²⁵I]galanin binding sites in the hypothalamusand amygdala. FIG. 4B-1: Total binding in the paraventricularhypothalamic nucleus (PVN), virtually all of which is removed by 60 nM[D-Trp²]galanin₍₁₋₂₉₎ (panel 3B). FIGS. 4B-3 and 4B-4: Binding in theventromedial hypothalamus (VMH), lateral hypothalamus (LH), and zonaincerta (ZI). In these regions, some [¹²⁵I]galanin binding remains afterincubation with 60 nM [D-Trp²]galanin₍₁₋₂₉₎ (FIG. 4B-4). FIGS. 4B-5 and4B-7: Total binding in the amygdala. After incubation with 60 nM[D-Trp²]galanin₍₁₋₂₉₎ (panels 5B and 6B), the binding is markedlyreduced in the piriform cortex (Pir), and to a lesser extent in themedial nucleus (Me), and central nucleus (Ce). However, the binding inthe nucleus of the lateral olfactory tract (LOT) is largely unaffected.FIG. 4C: FIGS. 4C-1 to 4C-6: Distribution of [¹²⁵I]galanin binding sitesin the anterior forebrain (FIGS. 4C-1 and 4C-2) and in the midbrain(FIGS. 4C-4 and 4C-5). FIGS. 4C-1 and 4C-2: In the lateral septum (LS)and insular cortex (CTX), much of the total binding (FIG. 4C-1) isremoved by 60 nM [D-Trp²]galanin₍₁₋₂₉₎ (FIG. 4C-2). FIGS. 4C-4 and 4C-5:Similarly, the total binding observed in the superior colliculus (SC),central gray (CG), and pontine reticular nucleus (PnO) (FIG. 4C-4) ismarkedly diminished by 60 nM [D-Trp²]galanin₍₁₋₂₉₎ (FIG. 4C-5). FIGS.4C-3 and 4C-6: Nonspecific binding observed in adjacent sections throughthe septum and midbrain. Arc, arcuate nucleus; Ce, central amygdaloidnucleus; CL, centrolateral thalamic nucleus; LOT, nucleus of the lateralolfactory tract; Me, medial amygdaloid nucleus; Pir, piriform cortex;PVN, paraventricular hypothalamic nucleus; SO, supraoptic nucleus; st,stria terminalis; VMH, ventromedial hypothalamic nucleus; ZI, zonaincerta.

FIG. 5. Reverse transcriptase PCR (RT-PCR) of rat GALR2 receptor mRNAfrom various brain regions. The blot was hybridized at high stringencywith an oligonucleotide probe corresponding to a portion of thepredicted V/VI loop of GALR2. Positive controls are indicated by +'s andrepresent plasmids containing the indicated inserts.

Size standards are indicated at the left in kilobases. Note theadditional hybridizing bands intermediate in size between theintron-containing and the intronless product.

FIGS. 6A-6B. Northern blot analysis of GALR2 receptor mRNA from variousrat brain regions. 6A. A Northern blot containing poly A⁺ RNA (˜5 μg)from six different rat brain regions was hybridized at high stringencywith a randomly primed radiolabeled fragment representing the entire ratGALR2 coding region (not including the intron). The autoradiogramrepresents a four day exposure and reveals a ˜1.8-2.0 kb transcript. 6B.The blot was reprobed with 1B15 (˜1 kb) to confirm that similar amountsof RNA were present in each lane.

FIGS. 7A-7B. Northern blot analysis of GALR2 receptor mRNA from variousrat tissues. 7A. A Northern blot containing poly A⁺ RNA (˜2 μg) fromeight different rat tissues was hybridized at high stringency with arandomly primed radiolabeled fragment representing the entire rat GALR2coding region (not including the intron). The autoradiogram represents afour day exposure and reveals a single ˜1.8-2.0 kb transcript. 7B. TheNorthern blot was reprobed for 1B15 (˜1 kb) to confirm that similaramounts of RNA were present in each lane. A second Northern blot (notshown) was also hybridized under the same conditions and showed similarresults (Table 3).

FIGS. 8A-8D. Rat GALR2 receptor autoradiography in COS-7 cellstransfected with GALR1 and GALR2 cDNAs. ¹²⁵I-[D-Trp²]Galanin₍₁₋₂₉₎ wastested as a selective radioligand for GALR2. Panels represent dark-fieldphotomicrographs (200×) of photoemulsion-dipped slides. 8A: Binding of 3nM ¹²⁵I-[D-Trp²]Galanin₍₁₋₂₉₎ to COS-7 cells transiently transfectedwith GALR2. Note positive binding to cells. 8B: Nonspecific binding of 6nM ¹²⁵I-[D-Trp²]Galanin₍₁₋₂₉₎ in the presence of 300 nM porcinegalanin₍₁₋₂₉₎ to COS-7 cells transiently transfected with GALR2. 8C:Binding of 6 nM ¹²⁵I-[D-Trp²]Galanin₍₁₋₂₉₎ to COS-7 cells transientlytransfected with GALR1. Note absence of binding to cell profiles; smallaccumulations of silver grains represent nonspecific nuclearassociation. 8D: Nonspecific binding of 6 nM ¹²⁵I-[D-Trp²]Galanin₍₁₋₂₉₎in the presence of 600 nM porcine galanin₍₁₋₂₉₎ to COS-7 cellstransiently transfected with GALR1.

FIGS. 9A-9B. Functional response mediated by LM(tk−) cells stablytransfected with the cDNA encoding the rat GALR2 receptor. 9A:Inhibition of cyclic AMP formation: cells were incubated with varyingconcentrations of porcine galanin₍₁₋₂₉₎, and 10 μM forskolin for 15 min.at 37° C. Data was normalized taking as 0% the basal levels of cyclicAMP (0.06±0.02 pmol/ml) and 100% the cAMP levels produced by forskolinin the absence of agonist (0.26±0.03 pmol/ml). Data is shown as mean ±standard error of the mean of four independent experiments. 9B:Phosphoinositide metabolism: cells were incubated for 18 hours in thepresence of 0.5 μCi [³H]myo-inositol. Eleven different concentrations ofporcine galanin₍₁₋₂₉₎ were added in the presence on 10 mM LiCl. Cellswere incubated for 1 hour at 37° C., and [³H]inositol phosphates wereisolated and measured.

FIG. 10. Nucleotide coding sequence of the human galanin GALR2 receptor(SEQ ID NO: 29), with partial 5′ and 3′ untranslated sequences. Start(ATG) and stop (TGA) codons are underlined.

FIG. 11. Deduced amino acid sequence of the human galanin GALR2 receptorencoded by the nucleotide sequence shown in FIG. 10 (Seq. I.D. No. 30).

FIGS. 12A-12C. 12A. Diagram of the intron-exon arrangement of the humanGALR2 receptor cDNA contained in plasmid BO29. Untranslated regions areindicated by dark hatched segments, and coding region is unmarked exceptfor light gray hatched segments indicating the location of thetransmembrane domains of the human GALR2 receptor. The black segmentindicates the location of the intron. 12B. Splice junction sequences ofthe human GALR2 receptor. 12C. Intron sequence of human GALR2 receptorcDNA contained in plasmid BO29 (SEQ ID NO: 31).

FIG. 13. Current response in an oocyte injected with 50 pg of GALR2mRNA. Holding potential was −80 mV.

FIG. 14. Autoradiograph demonstrating hybridization of radiolabeledrGalR2 probe to RNA extracted from rat. The lower band (arrow)represents mRNA coding for the rat GALR2 extracted from tissue indicatedat the bottom of the gel. RNA coding for the rat GALR2 is present in:the heart, kidney, hypothalamus, hippocampus, amygdala, spinal cord, andcerebellum. mRNA coding for the rat GALR2 was not detected in RNAextracted from striated muscle or liver.

FIGS. 15A-15D. Amino acid sequence alignment of the rat GALR2 receptor(top row) (SEQ ID NO: 8), human GALR2 receptor (middle row) (SEQ ID NO:29) and rat GALR1 receptor (bottom row) (SEQ ID NO: 32). Transmembranedomains (TM 1-7) are indicated by brackets above the sequence.

FIGS. 16A-16D. Galanin-mediated stimulation of phosphatidylinositolturnover and cyclic AMP inhibition in CHO cells expressing the rat GALR1and GALR2 receptors. 16A. CHO cell lines expressing the rat GALR1 (24pmol/mg protein, ▪) or the rat GALR2 (0.5 pmol/mg protein, ) receptorswere evaluated for galanin-dependent inhibition of forskolin-stimulatedcAMP accumulation by radioimmunoassay. 16B. The effect of 1 μM porcinegalanin on forskolin-stimulated cAMP accumulation was measured in CHOcells expressing the rat GALR1 receptor. Cells were incubated for 18 hrsin the presence (PTX) or absence (Control) of 100 ng/ml pertussis toxin.16C. The same CHO cell lines expressing rat GALR1 (▪) or rat GALR2 ()receptors were evaluated for galanin-dependent stimulation of inositolphosphate accumulation after an 18 hr incubation with [³H]myoinositol.16D. The effect of 1 μM porcine galanin on the release of [³H]inositolphosphates was measured in CHO cells expressing the rat GALR2 receptorincubated for 18 hrs in the presence (PTX), or absence (Control) of 100ng/ml pertussis toxin. Values represent the mean ± SEM from threedeterminations. Data shown are representative of three or moreindependent experiments.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this application, the following standard abbreviations areused to indicate specific nucleotide bases:

C = cytosine A = adenine T = thymine G = guanine

Furthermore, the term “agonist” is used throughout this application toindicate any peptide or non-peptidyl compound which increases theactivity of any of the receptors of the subject invention. The term“antagonist” is used throughout this application to indicate any peptideor non-peptidyl compound which decreases the activity of any of thereceptors of the subject invention.

The activity of a G-protein coupled receptor such as a galanin receptormay be measured using any of a variety of functional assays in whichactivation of the receptor in question results in an observable changein the level of some second messenger system, including but not limitedto adenylate cyclase, calcium mobilization, arachidonic acid release,ion channel activity, inositol phospholipid hydrolysis or guanylylcyclase. Heterologous expression systems utilizing appropriate hostcells to express the nucleic acid of the subject invention are used toobtain the desired second messenger coupling. Receptor activity may alsobe assayed in an oocyte expression system.

This invention provides an isolated nucleic acid encoding a vertebrateGALR2 receptor. In a separate embodiment, the nucleic acid encodes amammalian GALR2 receptor. In another embodiment, the nucleic acidencodes a rat GALR2 receptor. In still another embodiment, the nucleicacid encodes a human GALR2 receptor.

This invention further provides nucleic acid which is degenerate withrespect to the DNA comprising the coding sequence of the plasmid K985.This invention also provides nucleic acid which is degenerate withrespect to the DNA comprising the coding sequence of the plasmid K1045.This invention further provides nucleic acid which is degenerate withrespect to any DNA encoding a GALR2 receptor. In one embodiment, thenucleic acid comprises a nucleotide sequence which is degenerate withrespect to the nucleotide sequence described in FIG. 1 (SEQ ID NO: 1),that is, a nucleotide sequence which is translated into the same aminoacid sequence. In another embodiment, the nucleic acid comprises anucleotide sequence which is degenerate with respect to the nucleotidesequence described in SEQ ID NO: 9.

In yet another embodiment, this invention further provides nucleic acidwhich is degenerate with respect to the DNA comprising the codingsequence of plasmid BO29. In an embodiment, the nucleic acid comprises anucleotide sequence which is degenerate with respect to the nucleotidesequence described in FIG. 10 (SEQ ID NO: 29), that is, a nucleotidesequence which is translated into the same amino acid sequence. Thisinvention also provides nucleic acid which is degenerate with respect tothe DNA comprising the coding sequence of the plasmid BO39.

The observation that both the human and rat GALR2 cDNAs contain at leastone intron raises the possibility that additional introns could exist incoding or non-coding regions. In addition, spliced form(s) of mRNA mayencode additional amino acids either upstream of the currently definedstarting methionine or within the coding region. Further, the existenceand use of alternative exons is possible, whereby the mRNA may encodedifferent amino acids within the region comprising the exon. Inaddition, single amino acid substitutions may arise via the mechanism ofRNA editing such that the amino acid sequence of the expressed proteinis different than that encoded by the original gene (Burns et al., 1996;Chu et al., 1996). Such variants may exhibit pharmacologic propertiesdiffering from the receptor encoded by the original gene.

This invention provides a splice variant of the GALR2 receptorsdisclosed herein. This invention further provides for alternatetranslation initiation sites and alternately spliced or edited variantsof nucleic acids encoding rat and human GALR2 receptors.

This invention also encompasses DNAs and cDNAs which encode amino acidsequences which differ from those of the GALR2 galanin receptor, butwhich should not produce phenotypic changes. Alternatively, thisinvention also encompasses DNAs, cDNAs, and RNAs which hybridize to theDNA, cDNA, and RNA of the subject invention. Hybridization methods arewell known to those of skill in the art.

The nucleic acids of the subject invention also include nucleic acidmolecules coding for polypeptide analogs, fragments or derivatives ofantigenic polypeptides which differ from naturally-occurring forms interms of the identity or location of one or more amino acid residues(deletion analogs containing less than all of the residues specified forthe protein, substitution analogs wherein one or more residues specifiedare replaced by other residues and addition analogs where in one or moreamino acid residues is added to a terminal or medial portion of thepolypeptides) and which share some or all properties ofnaturally-occurring forms. These molecules include: the incorporation ofcodons “preferred” for expression by selected non-mammalian hosts; theprovision of sites for cleavage by restriction endonuclease enzymes; andthe provision of additional initial, terminal or intermediate DNAsequences that facilitate construction of readily expressed vectors.

G-protein coupled receptors such as the GALR2 receptors of the presentinvention are characterized by the ability of an agonist to promote theformation of a high-affinity ternary complex between the agonist, thereceptor, and an intracellular G-protein. This complex is formed in thepresence of physiological concentrations of GTP, and results in thedissociation of the alpha subunit of the G protein from the beta andgamma subunits of the G protein, which further results in a functionalresponse, i.e., activation of downstream effectors such as adenylylcyclase or phospholipase C. This high-affinity complex is transient evenin the presence of GTP, so that if the complex is destabilized, theaffinity of the receptor for agonists is reduced. Thus, if a receptor isnot optimally coupled to G protein under the conditions of an assay, anagonist will bind to the receptor with low affinity. In contrast, theaffinity of the receptor for an antagonist is normally not significantlyaffected by the presence or absence of G protein. Functional assays maybe used to determine whether a compound binds to the receptor, but maybe more time-consuming or difficult to perform than a binding assay.Therefore, it may desirable to produce a receptor which will bind toagonists with high affinity in a binding assay. Examples of modifiedreceptors which bind agonists with high affinity are disclosed in WO96/14331, which describes neuropeptide Y receptors modified in the thirdintracellular domain. The modifications may include deletions of 6-13amino acids in the third intracellular loop. Such deletions preferablyend immediately before the polar or charged residue at the beginning ofhelix six. In one embodiment, the deleted amino acids are at the carboxyterminal portion of the third intracellular domain. Such modifiedreceptors may be produced using methods well-known in the art such assite-directed mutagenesis or recombinant techniques using restrictionenzymes.

The modified receptors of this invention may be transfected into cellseither transiently or stably using methods well-known in the art,examples of which are disclosed herein. This invention also provides forbinding assays using the modified receptors, in which the receptor isexpressed either transiently or in stable cell lines. This inventionfurther provides for a compound identified using a modified receptor ina binding assay such as the binding assays described herein.

The nucleic acids described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide and as products for the large scale synthesis of thepolypeptide by a variety of recombinant techniques. The nucleic acidmolecule is useful for generating new cloning and expression vectors,transformed and transfected prokaryotic and eukaryotic host cells, andnew and useful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

This invention provides an isolated nucleic acid encoding a GALR2galanin receptor. This invention further provides a recombinant nucleicacid encoding a GALR2 galanin receptor.

In one embodiment of this invention the isolated nucleic acid is DNA. Inan embodiment, the DNA is cDNA. In another embodiment, the DNA isgenomic DNA. In still another embodiment, the nucleic acid molecule isRNA. In yet another embodiment of the present invention the nucleic acidmolecule is mRNA. Methods for production and manipulation of nucleicacid molecules are well known in the art.

In an embodiment, the galanin receptor is a vertebrate or a mammalianGALR2 receptor. In another embodiment, the galanin receptor is a ratGALR2 receptor. In another embodiment, the galanin receptor is a humanGALR2 receptor. In an embodiment, the isolated nucleic acid encodes areceptor characterized by an amino acid sequence in the transmembraneregion, which has a homology of 60% or higher to the amino acid sequencein the transmembrane region of the rat galanin GALR2 receptor and ahomology of less than 60% to the amino acid sequence in thetransmembrane region of any GALR1 receptor. In one embodiment, the GALR2receptor is a rat GALR2 receptor. In another embodiment, the GALR2receptor is a human GALR2 receptor.

In one embodiment, the GALR2 receptor has substantially the same aminoacid sequence as the amino acid sequence encoded by plasmid K985 (ATCCAccession No. 97426). In another embodiment, the GALR2 receptor has theamino acid sequence encoded by the plasmid K985. In still anotherembodiment, the GALR2 receptor has substantially the same amino acidsequence as the amino acid sequence encoded by the plasmid K1045 (ATCCAccession No. 97778). In yet another embodiment, the GALR2 receptor hasthe amino acid sequence encoded by the plasmid K1045. Plasmid K1045comprises an intronless cDNA encoding the rat GALR2 receptor. PlasmidK1045 is further characterized by its lack of native 5′ or 3′untranslated sequences, such that the plasmid contains only theregulatory elements necessary for expression in mammalian cells (e.g.,Kozak consensus sequence) and the coding sequence of the GALR2 receptor.

This invention provides an isolated nucleic acid encoding a GALR2receptor having substantially the same amino acid sequence as shown inFIG. 2. In one embodiment, the nucleic acid is DNA. This inventionfurther provides an isolated nucleic acid encoding a rat GALR2 receptorhaving the amino acid sequence shown in FIG. 2. In another embodiment,the nucleic acid comprises at least an intron. In yet anotherembodiment, the intron comprises a fragment of the intron sequence shownin FIG. 3C (SEQ ID NO: 9). In still another embodiment, the nucleic acidcomprises alternately spliced nucleic acid transcribed from the nucleicacid contained in plasmid K985. In one embodiment, the alternatelyspliced nucleic acid is mRNA transcribed from DNA encoding a galaninreceptor.

In one embodiment, the human GALR2 receptor has substantially the sameamino acid sequence as the amino acid sequence encoded by plasmid BO29(ATCC Accession No. 97735). In yet another embodiment, the human GALR2receptor has the amino acid sequence encoded by the plasmid BO29. Inanother embodiment, the nucleic acid encoding the human GALR2 receptorcomprises an intron. In still another embodiment, the nucleic acidencoding the human GALR2 receptor comprises alternately spliced nucleicacid transcribed from the nucleic acid contained in plasmid BO29. Instill another embodiment, the human GALR2 receptor has substantially thesame amino acid sequence as the amino acid sequence encoded by plasmidBO39 (ATCC Accession No. 97851). In another embodiment, the human GALR2receptor has the amino acid sequence encoded by the plasmid BO39.Plasmid BO39 comprises an intronless cDNA encoding the human GALR2receptor. This invention provides an isolated nucleic acid encoding aGALR2 receptor having substantially the same amino acid sequence asshown in FIG. 11 (SEQ ID NO: 30). In one embodiment, the nucleic acid isDNA. This invention further provides an isolated nucleic acid encoding ahuman GALR2 receptor having the amino acid sequence shown in FIG. 11.

This invention provides an isolated nucleic acid encoding a modifiedGALR2 receptor, which differs from a GALR2 receptor by having an aminoacid(s) deletion, replacement or addition in the third intracellulardomain. In one embodiment, the modified GALR2 receptor differs by havinga deletion in the third intracellular domain. In another embodiment, themodified GALR2 receptor differs by having an amino acid replacement oraddition to the third intracellular domain.

This invention also provides an isolated galanin GALR2 receptor protein.In one embodiment, the GALR2 receptor protein has the same orsubstantially the same amino acid sequence as the amino acid sequenceencoded by plasmid K985. In another embodiment, the GALR2 receptorprotein has the same or substantially the same amino acid sequence asthe amino acid sequence encoded by plasmid K1045. In one embodiment, theGALR2 receptor protein has the same or substantially the same amino acidsequence as shown in FIG. 2. In another embodiment, the GALR2 receptorhas the amino acid sequence shown in FIG. 2. In still anotherembodiment, the GALR2 receptor protein has the same or substantially thesame amino acid sequence as the amino acid sequence encoded by plasmidBO29. In still another embodiment, the GALR2 receptor protein has thesame or substantially the same amino acid sequence as the amino acidsequence encoded by plasmid BO39. In an embodiment, the GALR2 receptorprotein has the same or substantially the same amino acid sequence asshown in FIG. 11. In another embodiment, the GALR2 receptor has theamino acid sequence shown in FIG. 11.

This invention provides a vector comprising the above-described nucleicacid molecule. In one embodiment of the present invention the nucleicacid encodes a rat GALR2 receptor. In a further embodiment of thepresent invention the nucleic acid encodes a human GALR2 receptor.

Vectors which comprise the isolated nucleic acid molecule describedhereinabove also are provided. Suitable vectors comprise, but are notlimited to, a plasmid or a virus. These vectors may be transformed intoa suitable host cell to form a host cell expression system for theproduction of a polypeptide having the biological activity of a galaninGALR2 receptor. Suitable host cells include, for example, neuronal cellssuch as the glial cell line C6, a Xenopus cell such as an oocyte ormelanophore cell, as well as numerous mammalian cells and non-neuronalcells.

This invention provides the above-described vector adapted forexpression in a bacterial cell which further comprises the regulatoryelements necessary for expression of the nucleic acid in the bacterialcell operatively linked to the nucleic acid encoding the GALR2 receptoras to permit expression thereof.

This invention provides the above-described vector adapted forexpression in a yeast cell which comprises the regulatory elementsnecessary for expression of the nucleic acid in the yeast celloperatively linked to the nucleic acid encoding the GALR2 receptor as topermit expression thereof.

This invention provides the above-described vector adapted forexpression in an insect cell which comprises the regulatory elementsnecessary for expression of the nucleic acid in the insect celloperatively linked to the nucleic acid encoding the GALR2 receptor as topermit expression thereof. In a still further embodiment, the vector isa baculovirus.

In one embodiment, the vector is adapted for expression in a mammaliancell which comprises the regulatory elements necessary for expression ofthe nucleic acid in the mammalian cell operatively linked to the nucleicacid encoding the mammalian GALR2 receptor as to permit expressionthereof.

In a further embodiment, the vector is adapted for expression in amammalian cell which comprises the regulatory elements necessary forexpression of the nucleic acid in the mammalian cell operatively linkedto the nucleic acid encoding the rat GALR2 receptor as to permitexpression thereof.

In a still further embodiment, the vector is a plasmid.

In another embodiment, the plasmid is adapted for expression in amammalian cell which comprises the regulatory elements necessary forexpression of the nucleic acid in the mammalian cell operatively linkedto the nucleic acid encoding the human GALR2 receptor as to permitexpression thereof.

This invention provides the above-described plasmid adapted forexpression in a mammalian cell which comprises the regulatory elementsnecessary for expression of nucleic acid in a mammalian cell operativelylinked to the nucleic acid encoding the mammalian GALR2 receptor as topermit expression thereof.

This invention provides a plasmid designated K985 (ATCC Accession No.97426) which comprises the regulatory elements necessary for expressionof DNA in a mammalian cell operatively linked to DNA encoding the GALR2galanin receptor so as to permit expression thereof.

The plasmid designated K985 was deposited on Jan. 24, 1996, with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treatyfor the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure and was accorded ATCC Accession No.97426.

This invention provides a plasmid designated K1045 (ATCC Accession No.97778) which comprises the regulatory elements necessary for expressionof DNA in a mammalian cell operatively linked to DNA encoding the GALR2galanin receptor so as to permit expression thereof.

The plasmid designated K1045 was deposited on Oct. 30, 1996, with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treatyfor the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure and was accorded ATCC Accession No.97426.

This invention provides a plasmid designated BO29 (ATCC Accession No.97735) which comprises the regulatory elements necessary for expressionof DNA in a mammalian cell operatively linked to DNA encoding the GALR2galanin receptor as to permit expression thereof.

The plasmid designated BO29 was deposited on Sep. 25, 1996, with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treatyfor the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure and was accorded ATCC Accession No.97735.

This invention provides a plasmid designated BO39 (ATCC Accession No.97851) which comprises the regulatory elements necessary for expressionof DNA in a mammalian cell operatively linked to DNA encoding the GALR2galanin receptor as to permit expression thereof.

The plasmid designated BO39 was deposited on Jan. 15, 1997, with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treatyfor the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure and was accorded ATCC Accession No.97851.

This invention further provides for any vector or plasmid whichcomprises modified untranslated sequences, which are beneficial forexpression in desired host cells or for use in binding or functionalassays. For example, a vector or plasmid with untranslated sequences ofvarying lengths may express differing amounts of the receptor dependingupon the host cell used. In an embodiment, the vector or plasmidcomprises the coding sequence of the GALR2 receptor and the regulatoryelements necessary for expression in the host cell.

This invention provides a cell comprising the above-identified plasmidor vector. This invention provides a eukaryotic cell comprising theabove-described plasmid or vector. This invention provides anon-mammalian cell comprising the above-described plasmid or vector.This invention also provides a mammalian cell comprising theabove-described plasmid or vector. In an embodiment the cell is aXenopus oocyte or melanophore cell. In an embodiment, the cell is aneuronal cell such as the glial cell line C6. In an embodiment, themammalian cell is non-neuronal in origin. In an embodiment, themammalian cell is a COS-7 cell. In another embodiment the mammalian cellis a Chinese hamster ovary (CHO) cell. In another embodiment, the cellis a mouse Y1 cell.

In still another embodiment, the mammalian cell is a 293 human embryonickidney cell. In still another embodiment, the mammalian cell is aNIH-3T3 cell. In another embodiment, the mammalian cell is an LM(tk−)cell. In still another embodiment, the mammalian cell is the LM(tk−)cell designated L-rGALR2-8. This cell line was deposited with the ATCCon Mar. 28, 1996, under the provisions of the Budapest Treaty for theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure, and was accorded ATCC Accession No.CRL-12074. In yet another embodiment, the mammalian cell is the LM(tk−)cell designated L-rGALR2I-4 (which comprises the intronless plasmidK1045). This cell line was deposited with the ATCC on Oct. 30, 1996,under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure, and was accorded ATCC Accession No. CRL-12223.

In another embodiment, the mammalian cell is the Chinese hamster ovary(CHO) cell designated C-rGalR2-79. C-rGalR2-79 expresses the rat GALR2receptor and comprises a plasmid containing the intron within the codingregion. This cell line was deposited with the ATCC on Jan. 15, 1997,under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure, and was accorded ATCC Accession No. CRL-12262.

In another embodiment, the mammalian cell is the Chinese hamster ovary(CHO) cell designated CHO-hGALR2-264. CHO-hGALR2-264 expresses the humanGALR2 receptor and comprises the plasmid BO39. This cell line wasdeposited with the ATCC on Jul. 22, 1997, under the provisions of theBudapest Treaty for the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure, and was accordedATCC Accession No. CRL-12379.

This invention also provides an insect cell comprising theabove-described vector. In an embodiment, the insect cell is an Sf9cell. In another embodiment, the insect cell is an Sf21 cell.

This invention provides a membrane preparation isolated from any of theabove-described cells.

This invention provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding the GALR2 receptor contained in plasmid K985.

This invention further provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding the GALR2 receptor contained in plasmid K1045.

This invention still further provides a nucleic acid probe comprising atleast 15 nucleotides, which probe specifically hybridizes with a nucleicacid encoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within (a) the nucleic acid sequencedescribed in FIG. 1 or (b) the reverse complement thereto. Thisinvention also provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding the GALR2 receptor contained in plasmid BO29. Thisinvention also provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding the GALR2 receptor contained in plasmid BO39.

This invention provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within (a) the nucleic acid sequenceshown in FIG. 10 (SEQ ID NO: 29) or (b) the reverse complement to thenucleic acid sequence shown in FIG. 10.

This invention provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within (a) the nucleic acid sequenceshown in FIG. 1 (SEQ ID NO: 7) or (b) the reverse complement to thenucleic acid sequence shown in FIG. 1 (SEQ ID NO: 7). In one embodiment,the nucleic acid encoding a GALR2 receptor comprises an intron, thesequence of which intron is described in FIG. 3 (SEQ ID NO: 9). Inanother embodiment, the nucleic acid encoding a GALR2 receptor comprisesan intron, the sequence of which intron is described in FIG. 12C (SEQ IDNO: 31).

This invention further provides a nucleic acid probe comprising anucleic acid molecule of at least 15 nucleotides which is complementaryto a unique fragment of the sequence of a nucleic acid molecule encodinga GALR2 receptor. This invention also provides a nucleic acid probecomprising a nucleic acid molecule of at least 15 nucleotides which iscomplementary to the antisense sequence of a unique fragment of thesequence of a nucleic acid molecule encoding a GALR2 receptor.

In one embodiment, the nucleic acid probe is DNA. In another embodimentthe nucleic acid probe is RNA. As used herein, the phrase “specificallyhybridizing” means the ability of a nucleic acid molecule to recognize anucleic acid sequence complementary to its own and to formdouble-helical segments through hydrogen bonding between complementarybase pairs.

This nucleic acid of at least 15 nucleotides capable of specificallyhybridizing with a sequence of a nucleic acid encoding the GALR2 galaninreceptors can be used as a probe. In a further embodiment of the presentinvention the nucleic acid probe comprises a nucleic acid molecule of atleast 15 nucleotides which is complementary to the antisense sequence ofa unique fragment of the sequence of a nucleic acid molecule encoding aGALR2 receptor. Nucleic acid probe technology is well known to thoseskilled in the art who will readily appreciate that such probes may varygreatly in length and may be labeled with a detectable label, such as aradioisotope or fluorescent dye, to facilitate detection of the probe.DNA probe molecules may be produced by insertion of a DNA molecule whichencodes the GALR2 receptor into suitable vectors, such as plasmids orbacteriophages, followed by transforming into suitable bacterial hostcells, replication in the transformed bacterial host cells andharvesting of the DNA probes, using methods well known in the art.Alternatively, probes may be generated chemically from DNA synthesizers.

RNA probes may be generated by inserting the DNA molecule which encodesthe GALR2 galanin receptor downstream of a bacteriophage promoter suchas T3, T7 or SP6. Large amounts of RNA probe may be produced byincubating the labeled nucleotides with the linearized fragment where itcontains an upstream promoter in the presence of the appropriate RNApolymerase.

This invention provides an antisense oligonucleotide having a sequencecapable of specifically hybridizing to mRNA encoding a GALR2 galaninreceptor, so as to prevent translation of the mRNA.

This invention provides an antisense oligonucleotide having a sequencecapable of specifically hybridizing to the genomic DNA molecule encodinga GALR2 receptor.

This invention provides an antisense oligonucleotide comprising chemicalanalogues of nucleotides or chemically modified nucleotides.

This invention provides an antibody capable of binding to a GALR2receptor. This invention also provides an antibody capable of binding toa rat GALR2 receptor.

This invention also provides an antibody capable of binding to a humanGALR2 receptor. In an embodiment, the human GALR2 has an amino acidsequence the same or substantially the same as an amino acid sequenceencoded by plasmid K985 or an amino acid sequence encoded by plasmidBO29. In another embodiment, the human GALR2 has an amino acid sequencethe same or substantially the same as an amino acid sequence encoded byplasmid BO39.

This invention provides an antibody capable of competitively inhibitingthe binding of the antibody to a GALR2 receptor. In one embodiment ofthe present invention the antibody is a monoclonal antibody.

This invention provides a monoclonal antibody directed to an epitope ofa GALR2 receptor, which epitope is present on the surface of a cellexpressing a GALR2 receptor.

This invention provides a pharmaceutical composition comprising anamount of the oligonucleotide effective to reduce activity of a GALR2receptor by passing through a cell membrane and binding specificallywith mRNA encoding a GALR2 receptor in the cell so as to prevent itstranslation and a pharmaceutically acceptable carrier capable of passingthrough a cell membrane. In one embodiment, the oligonucleotide iscoupled to a substance which inactivates mRNA. In another embodiment,the substance which inactivates mRNA is a ribozyme.

This invention provides the above-described pharmaceutical composition,wherein the pharmaceutically acceptable carrier capable of passingthrough a cell membrane comprises a structure which binds to a receptorspecific for a selected cell type and is thereby taken up by cells ofthe selected cell type.

This invention provides a pharmaceutical composition comprising anamount of an antagonist effective to reduce the activity of a GALR2receptor and a pharmaceutically acceptable carrier.

This invention provides a pharmaceutical composition comprising anamount of an agonist effective to increase activity of a GALR2 receptorand a pharmaceutically acceptable carrier.

This invention provides the above-described pharmaceutical compositionwhich comprises an amount of the antibody effective to block binding ofa ligand to the GALR2 receptor and a pharmaceutically acceptablecarrier.

As used herein, “pharmaceutically acceptable carriers” means any of thestandard pharmaceutically acceptable carriers. Examples include, but arenot limited to, phosphate buffered saline, physiological saline, waterand emulsions, such as oil/water emulsions.

This invention provides a transgenic nonhuman mammal expressing DNAencoding a GALR2 receptor.

This invention provides a transgenic nonhuman mammal comprising ahomologous recombination knockout of the native GALR2 receptor.

This invention provides a transgenic nonhuman mammal whose genomecomprises antisense DNA complementary to DNA encoding a GALR2 receptorso placed as to be transcribed into antisense mRNA which iscomplementary to mRNA encoding a GALR2 receptor and which hybridizes tomRNA encoding a GALR2 receptor thereby reducing its translation.

This invention provides the above-described transgenic nonhuman mammal,wherein the DNA encoding a GALR2 receptor additionally comprises aninducible promoter.

This invention provides the transgenic nonhuman mammal, wherein the DNAencoding a GALR2 receptor additionally comprises tissue specificregulatory elements.

In an embodiment, the transgenic nonhuman mammal is a mouse.

Animal model systems which elucidate the physiological and behavioralroles of GALR2 receptor are produced by creating transgenic animals inwhich the activity of the GALR2 receptor is either increased ordecreased, or the amino acid sequence of the expressed GALR2 receptor isaltered, by a variety of techniques. Examples of these techniquesinclude, but are not limited to: 1) Insertion of normal or mutantversions of DNA encoding a GALR2 receptor, by microinjection,electroporation, retroviral transfection or other means well known tothose skilled in the art, into appropriate fertilized embryos in orderto produce a transgenic animal or 2) Homologous recombination of mutantor normal, human or animal versions of these genes with the native genelocus in transgenic animals to alter the regulation of expression or thestructure of these GALR2 receptor sequences. The technique of homologousrecombination is well known in the art. It replaces the native gene withthe inserted gene and so is useful for producing an animal that cannotexpress native GALR2 receptors but does express, for example, aninserted mutant GALR2 receptor, which has replaced the native GALR2receptor in the animal's genome by recombination, resulting inunderexpression of the transporter. Microinjection adds genes to thegenome, but does not remove them, and so is useful for producing ananimal which expresses its own and added GALR2 receptors, resulting inoverexpression of the GALR2 receptors.

One means available for producing a transgenic animal, with a mouse asan example, is as follows: Female mice are mated, and the resultingfertilized eggs are dissected out of their oviducts. The eggs are storedin an appropriate medium such as M2 medium. DNA or cDNA encoding a GALR2receptor is purified from a vector by methods well known in the art.Inducible promoters may be fused with the coding region of the DNA toprovide an experimental means to regulate expression of the trans-gene.Alternatively, or in addition, tissue specific regulatory elements maybe fused with the coding region to permit tissue-specific expression ofthe trans-gene. The DNA, in an appropriately buffered solution, is putinto a microinjection needle (which may be made from capillary tubingusing a pipet puller) and the egg to be injected is put in a depressionslide. The needle is inserted into the pronucleus of the egg, and theDNA solution is injected. The injected egg is then transferred into theoviduct of a pseudopregnant mouse (a mouse stimulated by the appropriatehormones to maintain pregnancy but which is not actually pregnant),where it proceeds to the uterus, implants, and develops to term. Asnoted above, microinjection is not the only method for inserting DNAinto the egg cell, and is used here only for exemplary purposes.

This invention provides a process for identifying a chemical compoundwhich specifically binds to a GALR2 receptor which comprises contactingcells containing DNA encoding and expressing on their cell surface theGALR2 receptor, wherein such cells do not normally express the GALR2receptor, with the compound under conditions suitable for binding, anddetecting specific binding of the chemical compound to the GALR2receptor.

This invention further provides a process for identifying a chemicalcompound which specifically binds to a GALR2 receptor which comprisescontacting a membrane fraction from a cell extract of cells containingDNA encoding and expressing on their cell surface the GALR2 receptor,wherein such cells do not normally express the GALR2 receptor, with thecompound under conditions suitable for binding, and detecting specificbinding of the chemical compound to the GALR2 receptor.

This invention also provides a method for determining whether a chemicalcompound can specifically bind to a GALR2 receptor which comprisescontacting cells transfected with and expressing DNA encoding the GALR2receptor with the compound under conditions permitting binding ofcompounds to such receptor, and detecting the presence of any suchcompound specifically bound to the GALR2 receptor, so as to therebydetermine whether the compound specifically binds to the GALR2 receptor.

This invention provides a method for determining whether a chemicalcompound can specifically bind to a GALR2 receptor which comprisespreparing a cell extract from cells transfected with and expressing DNAencoding the GALR2 receptor, isolating a membrane fraction from the cellextract, contacting the membrane fraction with the compound underconditions permitting binding of compounds to such receptor, anddetecting the presence of the compound specifically bound to the GALR2receptor, so as to thereby determine whether the compound specificallybinds to the GALR2 receptor.

In one embodiment, the GALR2 receptor is a mammalian GALR2 receptor. Inanother embodiment, the GALR2 receptor is a rat GALR2 receptor. In stillanother embodiment, the GALR2 receptor has the same or substantially thesame amino acid sequence as that encoded by plasmid K985, or plasmidK1045. In still another embodiment, the GALR2 receptor has the same orsubstantially the same amino acid sequence as the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 8). In yet another embodiment, the GALR2receptor has the amino acid sequence shown in FIG. 2 (SEQ ID NO: 8).

In another embodiment, the GALR2 receptor is a human GALR2 receptor. Instill another embodiment, the human GALR2 receptor has the same orsubstantially the same amino acid sequence as the amino acid sequenceencoded by plasmid BO29 or plasmid BO39. In yet another embodiment, theGALR2 receptor has the same or substantially the same amino acidsequence as the amino acid sequence shown in FIG. 11 (SEQ ID NO: 30). Inanother embodiment, the GALR2 receptor has the amino acid sequence shownin FIG. 11 (SEQ ID NO: 30).

This invention provides a process for determining whether a chemicalcompound is a GALR2 receptor agonist which comprises contacting cellstransfected with and expressing DNA encoding the GALR2 receptor with thecompound under conditions permitting the activation of the GALR2receptor, and detecting an increase in GALR2 receptor activity, so as tothereby determine whether the compound is a GALR2 receptor agonist.

This invention provides a process for determining whether a chemicalcompound is a GALR2 receptor agonist which comprises preparing a cellextract from cells transfected with and expressing DNA encoding theGALR2. receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with the compound under conditionspermitting the activation of the GALR2 receptor, and detecting anincrease in GALR2 receptor activity, so as to thereby determine whetherthe compound is a GALR2 receptor agonist.

This invention provides a process for determining whether a chemicalcompound is a GALR2 receptor agonist which comprises preparing a cellextract from cells transfected with and expressing DNA encoding theGALR2 receptor, isolating a membrane fraction from the cell extract,separately contacting the membrane fraction with both the chemicalcompound and GTPγS, and with only GTPγS, under conditions permitting theactivation of the GALR2 receptor, and detecting GTPγS binding to themembrane fraction, an increase in GTPγS binding in the presence of thecompound indicating that the chemical compound activates the GALR2receptor.

In one embodiment, the GALR2 receptor is a mammalian GALR2 receptor. Inanother embodiment, the GALR2 receptor is a rat GALR2 receptor. In stillanother embodiment, the GALR2 receptor has the same or substantially thesame amino acid sequence as that encoded by plasmid K985, or plasmidK1045. In still another embodiment, the GALR2 receptor has the same orsubstantially the same amino acid sequence as the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 8). In yet another embodiment, the GALR2receptor has the amino acid sequence shown in FIG. 2 (SEQ ID NO: 8).

In another embodiment, the GALR2 receptor is a human GALR2 receptor. Instill another embodiment, the human GALR2 receptor has the same orsubstantially the same amino acid sequence as the amino acid sequenceencoded by plasmid BO29 or plasmid BO39. In yet another embodiment, theGALR2 receptor has the same or substantially the same amino acidsequence as the amino acid sequence shown in FIG. 11 (SEQ ID NO: 30). Inanother embodiment, the GALR2 receptor has the amino acid sequence shownin FIG. 11 (SEQ ID NO: 30).

This invention provides a process for determining whether a chemicalcompound is a GALR2 receptor antagonist which comprises contacting cellstransfected with and expressing DNA encoding the GALR2 receptor with thecompound in the presence of a known GALR2 receptor agonist, such asgalanin, under conditions permitting the activation of the GALR2receptor, and detecting a decrease in GALR2 receptor activity, so as tothereby determine whether the compound is a GALR2 receptor antagonist.

This invention provides a process for determining whether a chemicalcompound is a GALR2 receptor antagonist which comprises preparing a cellextract from cells transfected with and expressing DNA encoding theGALR2 receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with the ligand in the presence of aknown GALR2 receptor agonist, such as galanin, under conditionspermitting the activation of the GALR2 receptor, and detecting adecrease in GALR2 receptor activity, so as to thereby determine whetherthe compound is a GALR2 receptor antagonist.

In one embodiment, the GALR2 receptor is a mammalian GALR2 receptor. Inanother embodiment, the GALR2 receptor is a rat GALR2 receptor. In stillanother embodiment, the GALR2 receptor has the same or substantially thesame amino acid sequence as that encoded by plasmid K985, or plasmidK1045. In still another embodiment, the GALR2 receptor has the same orsubstantially the same amino acid sequence as the amino acid sequenceshown in FIG. 2 (SEQ ID NO: 8). In yet another embodiment, the GALR2receptor has the amino acid sequence shown in FIG. 2 (SEQ ID NO: 8).

In another embodiment, the GALR2 receptor is a human GALR2 receptor. Instill another embodiment, the human GALR2 receptor has the same orsubstantially the same amino acid sequence as the amino acid sequenceencoded by plasmid BO29 or plasmid BO39. In yet another embodiment, theGALR2 receptor has the same or substantially the same amino acidsequence as the amino acid sequence shown in FIG. 11 (SEQ ID NO: 30). Inanother embodiment, the GALR2 receptor has the amino acid sequence shownin FIG. 11 (SEQ ID NO: 30).

In an embodiment of the above-described methods, the cell is an insectcell. In another embodiment, the cell is a mammalian cell. In a furtherembodiment, the cell is non-neuronal in origin. In still furtherembodiments, the non-neuronal cell is a COS-7 cell, 293 human embryonickidney cell, NIH-3T3 cell, a CHO cell, or LM(tk−) cell. In yet anotherembodiment of any of the processes of this invention the cell is theLM(tk−) cell L-rGALR2-8 (ATCC Accession No. CRL-12074), the LM(tk−) cellL-rGALR2I-4 (ATCC Accession No. CRL-12223, or the CHO cell C-rGalR2-79(ATCC Accession No. CRL-12262). In another embodiment of this inventionthe cell is the CHO cell CHO-hGALR2-264 (ATCC Accession No. CRL-).

In any of the above-described processes, receptor activity may bemeasured by assaying the binding of GTP gamma S (GTPγS) to membranes.GTPγS binding precedes the second messenger response of a G-proteincoupled receptor such as the GALR2 receptors of the present invention,providing a means of measuring activation of a receptor which isindependent of second messenger responses.

This invention provides a compound determined by the above-describedprocesses. In one embodiment of the above-described processes, thecompound is not previously known to bind to a GALR2 receptor.

This invention provides a GALR2 agonist determined by theabove-described processes. This invention also provides a GALR2antagonist determined by the above-described processes.

This invention provides a pharmaceutical composition which comprises anamount of a GALR2 receptor agonist effective to increase activity of aGALR2 receptor and a pharmaceutically acceptable carrier.

This invention provides a pharmaceutical composition which comprises anamount of a GALR2 receptor antagonist effective to reduce activity of aGALR2 receptor and a pharmaceutically acceptable carrier.

In further embodiments of the above-described processes, the agonist orantagonist is not previously known to bind to a GALR2 receptor.

This invention provides a process involving competitive binding foridentifying a chemical compound which specifically binds to a GALR2receptor, which comprises separately contacting cells expressing ontheir cell surface the GALR2 receptor, wherein such cells do notnormally express the GALR2 receptor, with both the chemical compound anda second chemical compound known to bind to the receptor, and with onlythe second chemical compound, under conditions suitable for binding ofboth compounds, and detecting specific binding of the chemical compoundto the GALR2 receptor, a decrease in the binding of the second chemicalcompound to the GALR2 receptor in the presence of the chemical compoundindicating that the chemical compound binds to the GALR2 receptor.

This invention further provides a process involving competitive bindingfor identifying a chemical compound which specifically binds to a humanGALR2 receptor, which comprises separately contacting a membranefraction from a cell extract of cells expressing on their cell surfacethe GALR2 receptor, wherein such cells do not normally express the GALR2receptor, with both the chemical compound and a second chemical compoundknown to bind to the receptor, and with only the second chemicalcompound, under conditions suitable for binding of both compounds, anddetecting specific binding of the chemical compound to the GALR2receptor, a decrease in the binding of the second chemical compound tothe GALR2 receptor in the presence of the chemical compound indicatingthat the chemical compound binds to the GALR2 receptor.

This invention further provides a process for determining whether achemical compound specifically binds to and activates a GALR2 receptor,which comprises contacting cells producing a second messenger responseand expressing on their cell surface the GALR2 receptor, wherein suchcells do not normally express the GALR2 receptor, with the chemicalcompound under conditions suitable for activation of the GALR2 receptor,and measuring the second messenger response in the presence and in theabsence of the chemical compound, a change in the second messengerresponse in the presence of the chemical compound indicating that thecompound activates the GALR2 receptor.

This invention further provides a process for determining whether achemical compound specifically binds to and activates a GALR2 receptor,which comprises contacting a membrane fraction from a cell extract ofcells producing a second messenger response and expressing on their cellsurface the GALR2 receptor, wherein such cells do not normally expressthe GALR2 receptor, with the chemical compound under conditions suitablefor activation of the GALR2 receptor, and measuring the second messengerresponse in the presence and in the absence of the chemical compound, achange in the second messenger response in the presence of the chemicalcompound indicating that the compound activates the GALR2 receptor.

In one embodiment of the above processes, the second messenger responsecomprises adenylate cyclase activity and the change in second messengerresponse is a decrease in adenylate cyclase activity. In one embodiment,adenylate cyclase activity is determined by measurement of cyclic AMPlevels.

In another embodiment of the above processes, the second messengerresponse comprises arachidonic acid release and the change in secondmessenger response is an increase in arachidonic acid levels.

In another embodiment of the above processes, the second messengerresponse comprises intracellular calcium levels and the change in secondmessenger response is an increase in intracellular calcium levels.

In a still further embodiment of the above processes, the secondmessenger response comprises inositol phospholipid hydrolysis and thechange in second messenger response is an increase in inositolphospholipid hydrolysis.

This invention further provides a process for determining whether achemical compound specifically binds to and inhibits activation of aGALR2 receptor, which comprises separately contacting cells producing asecond messenger response and expressing on their cell surface the GALR2receptor, wherein such cells do not normally express the GALR2 receptor,with both the chemical compound and a second chemical compound known toactivate the GALR2 receptor, and with only the second compound, underconditions suitable for activation of the GALR2 receptor, and measuringthe second messenger response in the presence of only the secondchemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change in the secondmessenger response in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the GALR2 receptor.

This invention further provides a process for determining whether achemical compound specifically binds to and inhibits activation of aGALR2 receptor, which comprises separately contacting a membranefraction from a cell extract of cells producing a second messengerresponse and expressing on their cell surface the GALR2 receptor,wherein such cells do not normally express the GALR2 receptor, with boththe chemical compound and a second chemical compound known to activatethe GALR2 receptor, and with only the second chemical compound, underconditions suitable for activation of the GALR2 receptor, and measuringthe second messenger response in the presence of only the secondchemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change in the secondmessenger response in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the GALR2 receptor.

In one embodiment of the above processes, the second messenger responsecomprises adenylate cyclase activity and the change in second messengerresponse is a smaller decrease in the level of adenylate cyclaseactivity in the presence of both the chemical compound and the secondchemical compound than in the presence of only the second chemicalcompound. In one embodiment, adenylate cyclase activity is determined bymeasurement of cyclic AMP levels.

In another embodiment of the above processes the second messengerresponse comprises arachidonic acid release, and the change in secondmessenger response is a smaller increase in arachidonic acid levels inthe presence of both the chemical compound and the second chemicalcompound than in the presence of only the second chemical compound.

In another embodiment of the above processes the second messengerresponse comprises intracellular calcium levels, and the change insecond messenger response is a smaller increase in intracellular calciumlevels in the presence of both the chemical compound and the secondchemical compound than in the presence of only the second chemicalcompound.

In yet another embodiment of the above processes, the second messengerresponse comprises inositol phospholipid hydrolysis, and the change insecond messenger response is a smaller increase in inositol phospholipidhydrolysis in the presence of both the chemical compound and the secondchemical compound than in the presence of only the second chemicalcompound.

In an embodiment of any of the above processes, the GALR2 receptor is amammalian GALR2 receptor. In another embodiment of the above processes,the GALR2 receptor is a rat GALR2 receptor or a human GALR2 receptor. Instill another embodiment of the above processes, the GALR2 receptor hasthe same or substantially the same amino acid sequence as encoded by theplasmid K985 ATCC Accession No. 97426), or plasmid K1045 (ATCC AccessionNo. 97778). In a still further embodiment of the above processes, theGALR2 receptor has the same or substantially the same amino acidsequence as that shown in FIG. 2 (SEQ ID NO: 8). In another embodimentof the above processes, the GALR2 receptor has the same or substantiallythe same amino acid sequence as the amino acid sequence encoded by theplasmid BO29 (ATCC Accession No. 97735) or the plasmid BO39 (ATCCAccession No. 97851). In a still further embodiment of the aboveprocesses, the GALR2 receptor has the same or substantially the sameamino acid sequence as that shown in FIG. 11 (SEQ ID NO: 30).

In an embodiment of any of the above processes, the cell is an insectcell. In another embodiment of any of the above processes, the cell is amammalian cell. In still further embodiments, the cell is nonneuronal inorigin.

In another embodiment of the above processes, the nonneuronal cell is aCOS-7 cell, Chinese hamster ovary (CHO) cell, 293 human embryonic kidneycell, NIH-3T3 cell, a mouse Y1 cell or LM(tk−) cell. In still furtherembodiments, nonneuronal cell is the LM(tk−) cell designated L-rGALR2-8(ATCC Accession No. CRL-12074), the LM(tk−) cell L-rGALR2I-4 (ATCCAccession No. CRL-12223, or the CHO cell C-rGalR2-79 (ATCC Accession No.CRL-12262). In another embodiment, the cell is the CHO cellCHO-hGALR2-264 (ATCC Accession No. CRL-).

This invention further provides a compound determined by any of theabove processes. In another embodiment, the compound is not previouslyknown to bind to a GALR2 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a GALR2 receptor to identify a compoundwhich specifically binds to the GALR2 receptor, which comprises (a)contacting cells transfected with and expressing DNA encoding the GALR2receptor with a compound known to bind specifically to the GALR2receptor; (b) contacting the preparation of step (a) with the pluralityof compounds not known to bind specifically to the GALR2 receptor, underconditions permitting binding of compounds known to bind the GALR2receptor; (c) determining whether the binding of the compound known tobind to the GALR2 receptor is reduced in the presence of the compounds,relative to the binding of the compound in the absence of the pluralityof compounds; and if so (d) separately determining the binding to theGALR2 receptor of each compound included in the plurality of compounds,so as to thereby identify the compound which specifically binds to theGALR2 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a GALR2 receptor to identify a compoundwhich specifically binds to the GALR2 receptor, which comprises (a)preparing a cell extract from cells transfected with and expressing DNAencoding the GALR2 receptor, isolating a membrane fraction from the cellextract, contacting the membrane fraction with a compound known to bindspecifically to the GALR2 receptor; (b) contacting the preparation ofstep (a) with the plurality of compounds not known to bind specificallyto the GALR2 receptor, under conditions permitting binding of compoundsknown to bind the GALR2 receptor; (c) determining whether the binding ofthe compound known to bind to the GALR2 receptor is reduced in thepresence of the compounds, relative to the binding of the compound inthe absence of the plurality of compounds; and if so (d) separatelydetermining the binding to the GALR2 receptor of each compound includedin the plurality of compounds, so as to thereby identify the compoundwhich specifically binds to the GALR2 receptor.

In an embodiment of the present invention the GALR2 receptor is amammalian GALR2 receptor. In one embodiment of the above-describedmethods, the GALR2 receptor is a rat GALR2 receptor. In anotherembodiment, the GALR2 receptor has the same or substantially the sameamino acid sequence as the amino acid sequence shown in FIG. 2 (SEQ IDNO: 8). In yet another embodiment, the GALR2 receptor has the amino acidsequence shown in FIG. 2 (SEQ ID NO: 8). In another embodiment, theGALR2 receptor is a human GALR2 receptor. In still another embodiment,the GALR2 receptor has the same or substantially the same amino acidsequence as the amino acid sequence encoded by plasmid BO29 or plasmidBO39. In another embodiment, the GALR2 receptor has the same orsubstantially the same amino acid sequence as the amino acid sequenceshown in FIG. 11 (SEQ ID NO: 30). In yet another embodiment, the GALR2receptor has the amino acid sequence shown in FIG. 11 (SEQ ID NO: 30).

This invention provides a method of screening a plurality of chemicalcompounds not known to activate a GALR2 receptor to identify a compoundwhich activates the GALR2 receptor which comprises (a) contacting cellstransfected with and expressing the GALR2 receptor with the plurality ofcompounds not known to activate the GALR2 receptor, under conditionspermitting activation of the GALR2 receptor; (b) determining whether theactivity of the GALR2 receptor is increased in the presence of thecompounds; and if so (c) separately determining whether the activationof the GALR2 receptor is increased by each compound included in theplurality of compounds, so as to thereby identify the compound whichactivates the GALR2 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to activate a GALR2 receptor to identify a compoundwhich activates the GALR2 receptor which comprises (a) preparing a cellextract from cells transfected with and expressing DNA encoding theGALR2 receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with the plurality of compounds notknown to activate the GALR2 receptor, under conditions permittingactivation of the GALR2 receptor; (b) determining whether the activityof the GALR2 receptor is increased in the presence of the compounds; andif so (c) separately determining whether the activation of the GALR2receptor is increased by each compound included in the plurality ofcompounds, so as to thereby identify the compound which activates theGALR2 receptor.

In an embodiment of the above-described methods, the GALR2 receptor is arat GALR2 receptor. In still another embodiment, the GALR2 receptor hasthe same or substantially the same amino acid sequence as the amino acidsequence shown in FIG. 2 (SEQ ID NO: 8). In yet another embodiment, theGALR2 receptor has the amino acid sequence shown in FIG. 2 (SEQ ID NO:8). In another embodiment, the GALR2 receptor is a human GALR2 receptor.In still another embodiment, the GALR2 receptor has the same orsubstantially the same amino acid sequence as the amino acid sequenceencoded by plasmid BO29 or plasmid BO39. In another embodiment, theGALR2 receptor has the same or substantially the same amino acidsequence as the amino acid sequence shown in FIG. 11 (SEQ ID NO: 30). Inyet another embodiment, the GALR2 receptor has the amino acid sequenceshown in FIG. 11 (SEQ ID NO: 30).

This invention provides a method of screening a plurality of chemicalcompounds not known to inhibit the activation of a GALR2 receptor toidentify a compound which inhibits the activation of the GALR2 receptor,which comprises (a) contacting cells transfected with and expressing theGALR2 receptor with the plurality of compounds in the presence of aknown GALR2 receptor agonist, under conditions permitting activation ofthe GALR2 receptor; (b) determining whether the activation of the GALR2receptor is reduced in the presence of the plurality of compounds,relative to the activation of the GALR2 receptor in the absence of theplurality of compounds; and if so (c) separately determining theinhibition of activation of the GALR2 receptor for each compoundincluded in the plurality of compounds, so as to thereby identify thecompound which inhibits the activation of the GALR2 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to inhibit the activation of a GALR2 receptor toidentify a compound which inhibits the activation of the GALR2 receptor,which comprises (a) preparing a cell extract from cells transfected withand expressing DNA encoding the GALR2 receptor, isolating a membranefraction from the cell extract, contacting the membrane fraction withthe plurality of compounds in the presence of a known GALR2 receptoragonist, under conditions permitting activation of the GALR2 receptor;(b) determining whether the activation of the GALR2 receptor is reducedin the presence of the plurality of compounds, relative to theactivation of the GALR2 receptor in the absence of the plurality ofcompounds; and if so (c) separately determining the inhibition ofactivation of the GALR2 receptor for each compound included in theplurality of compounds, so as to thereby identify the compound whichinhibits the activation of the GALR2 receptor.

In an embodiment of the above-described methods, the GALR2 receptor is arat GALR2 receptor. In another embodiment, the GALR2 receptor has thesame or substantially the same amino acid sequence as the amino acidsequence shown in FIG. 2 (SEQ ID NO: 8). In yet another embodiment, theGALR2 receptor has the amino acid sequence shown in FIG. 2 (SEQ ID NO:8). In another embodiment, the GALR2 receptor is a human GALR2 receptor.In still another embodiment, the GALR2 receptor has the same orsubstantially the same amino acid sequence as the amino acid sequenceencoded by plasmid BO29 or plasmid BO39. In another embodiment, theGALR2 receptor has the same or substantially the same amino acidsequence as the amino acid sequence shown in FIG. 11 (SEQ ID NO: 30). Inyet another embodiment, the GALR2 receptor has the amino acid sequenceshown in FIG. 11 (SEQ ID NO: 30).

In one embodiment of any of the above-described methods, the activationof the GALR2 receptor is determined by a second messenger assay. In anembodiment, the second messenger assay measures adenylate cyclaseactivity. In other embodiments, the second messenger is cyclic AMP,intracellular calcium, or arachidonic acid, an inositol phopholipid orphosphoinositol lipid metabolite.

In one embodiment, receptor activity may be measured by assaying thebinding of GTP gamma S (GTPγS) to membranes. In another embodiment,receptor activity may be measured by assaying changes in MAP kinasephosphorylation.

This invention further provides a method of measuring GALR2 receptoractivation in an oocyte expression system such as a Xenopus oocyte ormelanophore. In an embodiment, receptor activation is determined bymeasurement of ion channel activity. In another embodiment, receptoractivation is measured by aequorin luminescence.

Expression of genes in Xenopus oocytes is well known in the art (A.Coleman, Transcription and Translation: A Practical Approach (B. D.Hanes, S. J. Higgins, eds., pp 271-302, IRL Press, Oxford, 1984; Y. Masuet al., Nature 329:21583-21586, 1994) and is performed usingmicroinjection of native mRNA or in vitro synthesized mRNA into frogoocytes. The preparation of in vitro synthesized mRNA can be performedby various standard techniques (J. Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1989) including using T7 polymerase with the mCAPRNA capping kit (Stratagene).

In a further embodiment of the invention, the cell is a mammalian cell.In another embodiment of the invention, the mammalian cell isnon-neuronal in origin. In still further embodiments of the invention,the non-neuronal cell is a COS-7 cell, a 293 human embryonic kidneycell, a mouse Y1 cell, a LM(tk−) cell, a CHO cell, or an NIH-3T3 cell.In an embodiment of the invention, the nonneuronal cell is the LM(tk−)cell designated L-rGALR2-8 (ATCC Accession No. CRL-12074), the LM(tk−)cell L-rGALR2I-4 (ATCC Accession No. CRL-12223, or the CHO cellC-rGalR2-79 (ATCC Accession No. CRL-12262). In another embodiment, thecell is the CHO cell CHO-hGALR2-264 (ATCC Accession No. CRL-).

This invention provides a pharmaceutical composition comprising acompound identified by the above-described methods and apharmaceutically acceptable carrier.

In an embodiment of the above-described methods, the cell isnon-neuronal in origin. In a further embodiment, the non-neuronal cellis a COS-7 cell, CHO cell, 293 human embryonic kidney cell, NIH-3T3cell, a mouse Y1 cell or LM(tk−) cell.

In one embodiment of the above-described methods, the compound is notpreviously known.

This invention provides a GALR2 receptor agonist detected by theabove-described methods. This invention provides a GALR2 receptorantagonist detected by the above-described methods. In an embodiment thecell is a non-mammalian cell, for example, a Xenopus oocyte ormelanophore. In another embodiment the cell is a neuronal cell, forexample, a glial cell line such as C6. In an embodiment, the cell isnon-neuronal in origin. In a further embodiment, the cell is a Cos-7 ora CHO cell, a 293 human embryonic kidney cell, an LM(tk−) cell or anNIH-3T3 cell. In an embodiment of the invention, the LM(tk−) cell is thecell designated L-rGALR2-8 (ATCC Accession No. CRL-12074), the LM(tk−)cell L-rGALR2I-4 (ATCC Accession No. CRL-12223, or the CHO cellC-rGalR2-79 (ATCC Accession No. CRL-12262). In another embodiment, thecell is the CHO cell CHO-hGALR2-264 (ATCC Accession No. CRL-).

This invention provides a pharmaceutical composition comprising a drugcandidate identified by the above-described methods and apharmaceutically acceptable carrier.

This invention provides a method of detecting expression of a GALR2receptor by detecting the presence of mRNA coding for the GALR2 receptorwhich comprises obtaining total mRNA from a cell or tissue sample andcontacting the mRNA so obtained with the above-described nucleic acidprobe under hybridizing conditions, detecting the presence of mRNAhybridized to the probe, and thereby detecting the expression of theGALR2 receptor by the cell or in the tissue.

This invention provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by administering to thesubject an amount of a GALR2 selective compound, effective to treat theabnormality. Abnormalities which may be treated include cognitivedisorder, pain, sensory disorder (olfactory, visual), motor coordinationabnormality, motion sickness, neuroendocrine disorders, sleep disorders,migraine, Parkinson's disease, hypertension, heart failure,convulsion/epilepsy, traumatic brain injury, diabetes, glaucoma,electrolyte imbalances, respiratory disorders (asthma, emphysema),depression, reproductive disorders, gastric and intestinal ulcers,gastroesophageal reflux disorder, gastric hypersecretion,gastrointestinal motility disorders (diarrhea), inflammation, immunedisorders, and anxiety. In one embodiment the compound is an agonist. Inanother embodiment the compound is an antagonist.

This invention provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by the inhibition of aGALR2 receptor which comprises administering to a subject an effectiveamount of the above-described pharmaceutical composition effective todecrease the activity of the GALR2 receptor in the subject, therebytreating the abnormality in the subject. In an embodiment, theabnormality is obesity. In another embodiment, the abnormality isbulimia.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by the activation of a GALR2receptor which comprises administering to a subject an effective amountof the above-described pharmaceutical composition effective to activatethe GALR2 receptor in the subject. In an embodiment, the abnormalcondition is anorexia.

In another embodiment, the compound binds selectively to a GALR2receptor. In yet another embodiment, the compound binds to the GALR2receptor with an affinity greater than ten-fold higher than the affinitywith which the compound binds to a GALR1 receptor. In a still furtherembodiment, the compound binds to the GALR2 receptor with an affinitygreater than ten-fold higher than the affinity with which the compoundbinds to a GALR3 receptor.

This invention provides a method of detecting the presence of a GALR2receptor on the surface of a cell which comprises contacting the cellwith the above-described antibody under conditions permitting binding ofthe antibody to the receptor, detecting the presence of the antibodybound to the cell, and thereby detecting the presence of a GALR2receptor on the surface of the cell.

This invention provides a method of determining the physiologicaleffects of varying levels of activity of GALR2 receptors which comprisesproducing a transgenic nonhuman mammal whose levels of GALR2 receptoractivity are varied by use of an inducible promoter which regulatesGALR2 receptor expression.

This invention provides a method of determining the physiologicaleffects of varying levels of activity of GALR2 receptors which comprisesproducing a panel of transgenic nonhuman mammals each expressing adifferent amount of GALR2 receptor.

This invention provides a method for identifying an antagonist capableof alleviating an abnormality wherein the abnormality is alleviated bydecreasing the activity of a GALR2 receptor comprising administering acompound to the above-described transgenic nonhuman mammal anddetermining whether the compound alleviates the physical and behavioralabnormalities displayed by the transgenic nonhuman mammal as a result ofoveractivity of a GALR2 receptor, the alleviation of the abnormalityidentifying the compound as an antagonist.

This invention provides an antagonist identified by the above-describedmethods. This invention provides a pharmaceutical composition comprisingan antagonist identified by the above-described methods and apharmaceutically acceptable carrier.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by decreasing the activity of aGALR2 receptor which comprises administering to a subject an effectiveamount of the above-described pharmaceutical composition, therebytreating the abnormality.

This invention provides a method for identifying an agonist capable ofalleviating an abnormality in a subject wherein the abnormality isalleviated by increasing the activity of a GALR2 receptor comprisingadministering a compound to a transgenic nonhuman mammal and determiningwhether the compound alleviates the physical and behavioralabnormalities displayed by the transgenic nonhuman mammal, thealleviation of the abnormality identifying the compound as an agonist.

This invention provides an agonist identified by the above-describedmethods.

This invention provides a pharmaceutical composition comprising anagonist identified by the above-described methods and a pharmaceuticallyacceptable carrier.

This invention provides a method for treating an abnormality in asubject wherein the abnormality is alleviated by increasing the activityof a GALR2 receptor which comprises administering to a subject aneffective amount of the above-described pharmaceutical composition,thereby treating the abnormality.

This invention provides a method for diagnosing a predisposition to adisorder associated with the activity of a specific human GALR2 receptorallele which comprises: (a) obtaining DNA of subjects suffering from thedisorder; (b) performing a restriction digest of the DNA with a panel ofrestriction enzymes; (c) electrophoretically separating the resultingDNA fragments on a sizing gel; (d) contacting the resulting gel with anucleic acid probe capable of specifically hybridizing with a uniquesequence included within the sequence of a nucleic acid moleculeencoding a human GALR2 receptor and labelled with a detectable marker;(e) detecting labelled bands which have hybridized to DNA encoding ahuman GALR2 receptor labelled with a detectable marker to create aunique band pattern specific to the DNA of subjects suffering from thedisorder; (f) preparing DNA obtained for diagnosis by steps a-e; and (g)comparing the unique band pattern specific to the DNA of subjectssuffering from the disorder from step e and the DNA obtained fordiagnosis from step f to determine whether the patterns are the same ordifferent and to diagnose thereby predisposition to the disorder if thepatterns are the same.

In an embodiment, a disorder associated with the activity of a specifichuman GALR2 receptor allele is diagnosed. In another embodiment, theabove-described method may be used to identify a population of patientshaving a specific GALR2 receptor allele, in which population thedisorder may be alleviated by administering to the subjects aGALR2-selective compound.

This invention provides a method of preparing the purified GALR2receptor which comprises: (a) inducing cells to express GALR2 receptor;(b) recovering the receptor from the induced cells; and (c) purifyingthe receptor so recovered.

This invention provides a method of preparing a purified GALR2 receptorwhich comprises: (a) inserting nucleic acid encoding the GALR2 receptorin a suitable vector; (b) introducing the resulting vector in a suitablehost cell; (c) placing the resulting cell in suitable conditionpermitting the production of the isolated GALR2 receptor; (d) recoveringthe receptor produced by the resulting cell; and (e) purifying thereceptor so recovered.

This invention provides a method of modifying feeding behavior of asubject which comprises administering to the subject an amount of acompound which is a galanin receptor agonist or antagonist effective toincrease or decrease the consumption of food by the subject so as tothereby modify feeding behavior of the subject. In one embodiment, thecompound is a GALR2 receptor antagonist and the amount is effective todecrease the consumption of food by the subject. In another embodimentthe compound is administered in combination with food.

In yet another embodiment the compound is a GALR2 receptor agonist andthe amount is effective to increase the consumption of food by thesubject. In a still further embodiment, the compound is administered incombination with food. In other embodiments the subject is a vertebrate,a mammal, a human or a canine.

In one embodiment, the compound binds selectively to a GALR2 receptor.In another embodiment, the compound binds to the GALR2 receptor with anaffinity greater than ten-fold higher than the affinity with which thecompound binds to a GALR1 receptor. In another embodiment, the compoundbinds to the GALR2 receptor with an affinity greater than ten-foldhigher than the affinity with which the compound binds to a GALR3receptor. In yet another embodiment, the compound binds to the GALR2receptor with an affinity greater than one hundred-fold higher than theaffinity with which the compound binds to a GALR1 receptor. In anotherembodiment, the compound binds to the GALR2 receptor with an affinitygreater than one hundred-fold higher than the affinity with which thecompound binds to a GALR3 receptor.

This invention provides a method for determining whether a chemicalcompound is a GALR2 antagonist which comprises: (a) administering to ananimal a GALR2 agonist and measuring the amount of food intake in theanimal; (b) administering to a second animal both the GALR2 agonist andthe chemical compound, and measuring the amount of food intake in thesecond animal; and (c) determining whether the amount of food intake isreduced in the presence of the chemical compound relative to the amountof food intake in the absence of the compound, so as to therebydetermine whether the compound is a GALR2 antagonist.

This invention further provides a method of screening a plurality ofchemical compounds to identify a chemical compound which is a GALR2antagonist which comprises: (a) administering to an animal a GALR2agonist and measuring the amount of food intake in the animal; (b)administering to a second animal the GALR2 agonist and at least onechemical compound of the plurality of compounds, and measuring theamount of food intake in the animal; (c) determining whether the amountof food intake is reduced in the presence of at least one chemicalcompound of the plurality of chemical compounds relative to the amountof food intake in the absence of at least one of the compounds, and ifso; (d) separately determining whether each chemical compound is a GALR2antagonist according to the method described above, so as to therebydetermine if the chemical compound is a GALR2 antagonist. In oneembodiment the GALR2 agonist is [D-Trp]₂-galanin₍₁₋₂₉₎. In anotherembodiment the animal is a non-human mammal. In a further embodiment,the animal is a rodent.

In one embodiment, the above process further comprises determiningwhether the compound selectively binds to the GALR2 receptor relative toanother galanin receptor. In another embodiment, the determinationwhether the compound selectively binds to the GALR2 receptor comprises:(a) determining the binding affinity of the compound for the GALR2receptor and for such other galanin receptor; and (b) comparing thebinding affinities so determined, the presence of a higher bindingaffinity for the GALR2 receptor than for such other galanin receptorindicating that the compound selectively binds to the GALR2 receptor. Inan embodiment, the other galanin receptor is a GALR1 receptor. Inanother embodiment, the other galanin receptor is a GALR3 receptor.

This invention provides a process for determining whether a compoundselectively activates the GALR2 receptor relative to another galaninreceptor.

This invention provides a process for determining whether a compoundselectively activates the GALR2 receptor relative to another galaninreceptor, wherein the determination whether the compound selectivelyactivates the GALR2 receptor comprises: (a) determining the potency ofthe compound for the GALR2 receptor and for such other galanin receptor;and (b) comparing the potencies so determined, the presence of a higherpotency for the GALR2 receptor than for such other galanin receptorindicating that the compound selectively activates the GALR2 receptor.In an embodiment, such other galanin receptor is a GALR1 receptor. Inanother embodiment, such other galanin receptor is a GALR3 receptor.

This invention provides a process for determining whether a compoundselectively inhibits the activation of the GALR2 receptor relative toanother galanin receptor.

This invention provides a process for determining whether a compoundselectively inhibits the activation of the GALR2 receptor relative toanother galanin recpetor, wherein the determination whether the compoundselectively inhibits the activation of the GALR2 receptor comprises: (a)determining the decrease in the potency of a known galanin receptoragonist for the GALR2 receptor in the presence of the compound, relativeto the potency of the agonist in the absence of the compound; (b)determining the decrease in the potency of the agonist for such othergalanin receptor in the presence of the compound, relative to thepotency of the agonist in the absence of the compound; and (c) comparingthe decrease in potencies so determined, the presence of a greaterdecrease in potency for the GALR2 receptor than for such other galaninreceptor indicating that the compound selectively inhibits theactivation of the GALR2 receptor. In an embodiment, such other galaninreceptor is a GALR1 receptor. In another embodiment, such other galaninreceptor is a GALR3 receptor.

This invention provides a method of decreasing feeding behavior of asubject which comprises administering a compound which is a GALR2receptor antagonist and a compound which is a Y5 receptor antagonist,the amount of such antagonists being effective to decrease the feedingbehavior of the subject. In one embodiment, the GALR2 antagonist and theY5 antagonist are administered in combination. In another embodiment,the GALR2 antagonist and the Y5 antagonist are administered once. Inanother embodiment, the GALR2 antagonist and the Y5 antagonist areadministered separately. In still another embodiment, the GALR2antagonist and the Y5 antagonist are administered once. In anotherembodiment, the galanin receptor antagonist is administered for about 1week to 2 weeks. In another embodiment, the Y5 receptor antagonist isadministered for about 1 week to 2 weeks.

In yet another embodiment, the GALR2 antagonist and the Y5 antagonistare administered alternately. In another embodiment, the GALR2antagonist and the Y5 antagonist are administered repeatedly. In a stillfurther embodiment, the galanin receptor antagonist is administered forabout 1 week to 2 weeks. In another embodiment, the Y5 receptorantagonist is administered for about 1 week to 2 weeks.

This invention also provides a method as described above, wherein thecompound is administered in a pharmaceutical composition comprising asustained release formulation.

This invention provides a method of decreasing nociception in a subjectwhich comprises administering to the subject an amount of a compoundwhich is a GALR2 receptor agonist effective to decrease nociception inthe subject.

This invention provides a method of treating pain in a subject whichcomprises administering to the subject an amount of a compound which isa GALR2 receptor agonist effective to treat pain in the subject.

This invention provides a method of treating Alzheimer's disease in asubject which comprises administering to the subject an amount of acompound which is a galanin receptor antagonist effective to treat thesubject's Alzheimer's disease. In one embodiment, the galanin receptorantagonist is a GALR2 receptor antagonist and the amount of the compoundis effective to treat the subject's Alzheimer's disease.

This invention provides a method of producing analgesia in a subjectwhich comprises administering to the subject an amount of a compoundwhich is a galanin receptor agonist effective to produce analgesia inthe subject.

In another embodiment, the galanin receptor agonist is a GALR2 receptoragonist and the amount of the compound is effective to produce analgesiain the subject.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

Experimental Details

Materials and Methods

Construction and Screening of a Rat Hypothalamus cDNA Library

Total RNA was prepared from rat hypothalami by a modification of theguanidine thiocyanate method (Chirgwin, 1979). Poly A⁺ RNA was purifiedusing a FastTrack kit (Invitrogen Corp., San Diego, Calif.). Doublestranded (ds) cDNA was synthesized from 4.6 μg of poly A⁺ RNA accordingto Gubler and Hoffman (1983) with minor modifications. The resultingcDNA was ligated to BstXI/EcoRI adaptors (Invitrogen Corp.) and theexcess adaptors removed by exclusion column chromatography. Highmolecular weight fractions of size-selected ds-cDNA were ligated inpEXJ.T7 (an Okayama and Berg expression vector modified from pcEXV(Miller & Germain, 1986) to contain BstXI and other additionalrestriction sites and a T7 promoter (Stratagene) and electroporated inE.coli MC 1061 (Gene Pulser, Biorad). A total of 3×10⁶ independentclones with a mean insert size of 2.2 kb were generated. The library wasplated on agar plates (Ampicillin selection) in 584 primary pools of˜5,000 independent clones. After 18 hours amplification, the bacteriafrom each pool were scraped, resuspended in 4 mL of LB media and 0.75 mLprocessed for plasmid purification (QIAwell-96 ultra, Qiagen,Inc.,Chatsworth, Calif.). Aliquots of each bacterial pool were stored at −85°C. in 20% glycerol.

To screen the library, COS-7 cells were plated in slide chambers(Lab-Tek) in Dulbecco's modified Eagle medium (DMEM) supplemented with10% calf serum, 100 U/mL of penicillin, 100 ug/mL streptomycin, 2 mML-glutamine (DMEM-C) and grown at 37° C. in a humidified 5% CO₂atmosphere for 24 hours before transfection. Cells were transfected withminiprep DNA prepared from the primary pools (˜4,500 cfu/pool) of therat hypothalamus cDNA library using a modification of the DEAE-dextranmethod (Warden & Thorne, 1968). Pools containing GALR1 were identifiedby PCR prior to screening and were omitted from the primary screen. Thegalanin binding assay was carried out after 48 hours. Cells were rinsedtwice with phosphate-buffered saline (PBS) then incubated with 1 nM¹²⁵I-porcine galanin (NEN; specific activity ˜2200 Ci/mmol) in 20 mMHEPES-NaOH, pH 7.4, containing 1.26 mM CaCl₂, 0.81 mM MgSO₄, 0.44 mMKH₂PO₄, 5.4 mM KCl, 10 mM NaCl, 0.1% BSA, and 0.1% bacitracin for onehour at room temperature. After rinsing and fixation in 2.5%glutaraldehyde, slides were rinsed in PBS, air-dried, and dipped inphotoemulsion (Kodak, NTB-2). After a 3-4 day exposure slides weredeveloped in Kodak D19 developer, fixed, and coverslipped (Aqua-Mount,Lerner Laboratories), then inspected for positive cells by brightfieldmicroscopy (Leitz Laborlux, 25× magnification). One pool with positivecells, (J126) was subdivided and rescreened repeatedly until a singlecolony was isolated that conferred galanin binding. The 3.8 kb cDNA ispreferably sequenced on both strands using Sequenase (US Biochemical,Cleveland, Ohio) according to the manufacturer. Nucleotide and peptidesequence analyses are performed using the Wisconsin Package (GCG,Genetics Computer group, Madison, Wis.) or PC/GENE (Intelligenetics,Mountain View, Calif.).

PCR Methodology

PCR reactions were carried out in 20 μL volumes using Taq Polymerase(Boehringer Mannheim, Indianapolis, Ind.) in a buffer containing 10 mMTris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂ 0.01% gelatin, 0.2 mM eachdNTP, and 1 μM each PCR primer. To prescreen library pools for GALR1,two GALR1 primer sets were used (KS-1177/1178 and KS-1311/1313, seebelow) to determine whether GALR1 was present in original bacterialstocks of each library pool. PCR was carried out for 40 cycles of 94°C./2 min, 68° C./2 min, 72° C./3 min. Pools positive for GALR1 by PCRwere eliminated from the library screen.

To confirm that the purified cDNA conferring galanin binding wasdistinct from GALR1, the isolated clone representing pool J126-10-334(K985) was subjected to PCR analysis using three GALRl primer setsrepresenting different regions of GALR1. The nucleotide sequences of theprimer sets are shown below:

KS-1177: 5′-TGG GCA ACA GCC TAG TGA TCA CCG-3′ (Seq. I.D. No. 1)Nucleotides 146-169 of human GALR1 coding region, forward primer.

KS-1178: 5′-CTG CTC CCA GCA GAA GGT CTG GTT-3′ (Seq. I.D. No. 2)Nucleotides 547-570 of human GALR1 coding region, reverse primer.

KS-1311: 5′-CCT CAG TGA AGG GAA TGG GAG CGA-3′ (Seq. I.D. No. 3)Nucleotides 21-44 of rat GALR1 coding region, forward primer.

KS-1313: 5′-CTC ATT GCA AAC ACG GCA CTT GAA CA-3′ (Seq. I.D. No. 4)Nucleotides 944-969 of rat GALR1 coding region, reverse primer.

KS-1447: 5′-CTT GCT TGT ACG CCT TCC GGA AGT-3′ (Seq. I.D. No. 5)Nucleotides 920-943 of rat GALR1 coding region, reverse primer.

KS-1448: 5′-GAG AAC TTC ATC ACG CTG GTG GTG-3′ (Seq. I.D. No. 6).Nucleotides 91-114 of rat GALR1 coding region, forward primer.

Generation of Human GALR2 PCR Product Human genomic DNA (1 μg; 12different lots from Promega and Clontech) were amplified in 50 μl PCRreaction mixtures using the Expand Long Template PCR System (as suppliedand described by the manufacturer, Boehringer Mannheim) and 1 μM ofprimers, using a program consisting of 40 cycles of 94° C. for 2 min,60° C. for 2 min, and 68° C. for 3 min, with a pre- and post-incubationof 95° C. for 5 min and 68° C. for 10 min, respectively. PCR primers forhGALR2 were designed against rGALR2 sequence: forward primer NS525 inthe fourth transmembrane domain, and reverse primer NS526 in the sixthtransmembrane domain. The PCR products were run on a 0.8% low-meltingagarose gel. The single ≈300 bp fragment from 3 different lots wereisolated, purified by phenol extraction and subjected to sequencingusing the T7 Sequenase PCR product sequencing kit (Amersham). Sequencewas analyzed using the Wisconsin Package (GCG, Genetics Computer Group,Madison, Wis.).

5′ and 3′ RACE Analysis of Human GALR2

5′ and 3′ RACE (rapid analysis of cDNA ends) were performed on humanbrain and human lung RNAs (Clontech), respectively, using a MarathoncDNA Amplification Kit (Clontech). Total RNA was poly A+ selected usinga FastTrack mRNA Isolation Kit (Invitrogen Corp., San Diego, Calif.).For 5′ RACE, double stranded (ds) cDNA was synthesized from 1 μg Poly A+RNA using BB 153, a reverse primer from the 5′ end of the sixthtransmembrane domain of hGALR2 from the PCR fragment described above.Adaptor ligation and nested PCR were performed according to the MarathoncDNA Amplification protocol using Advantage KlenTaq Polymerase(Clontech). The initial PCR reaction was performed on 1 μl of a 50 folddilution of the ligated cDNA using the supplier's Adaptor Primer 1 andBB 154, a reverse primer from the fifth transmembrane domain of thehGALR2 PCR product above. One μl of this initial PCR reaction wasre-amplified using the Adaptor Primer 2 and NS 563, a reverse primerjust upstream from BB154. The conditions for PCR were 30 sec at 94° C.,4 min at 72° C. for 5 cycles, 30 sec at 94° C., 4 min at 70° C. for 5cycles, 20 sec at 94° C., 4 min at 68° C. for 25 cycles, with a pre- andpost-incubation of 1 min at 94° C. and 7 min at 68° C. respectively. A600 base pair fragment from the nested PCR was isolated from a 1% TAEgel using a GENECLEAN III kit (BIO 101, Vista, Calif.) and sequencedusing AmpliTaq DNA Polymerase, FS (Perkin Elmer). The sequence was runon an ABI PRISM 377 DNA Sequencer and analyzed using the WisconsinPackage (GCG, Genetics Computer Group, Madison, Wis.). For 3′ RACE,double stranded (ds) cDNA was synthesized from 1 μg Poly A+ RNA usingthe cDNA synthesis primer CDS supplied with the Marathon cDNAAmplification Kit (Clontech). PCR conditions for 3′ RACE were similar to5′ RACE except that BB166 and BB167, forward primers from the fifthtransmembrane domain of the hGALR2 PCR fragment described above, wereused in place of BB154 and NS563, respectively. A 500 base pair fragmentfrom the nested PCR was isolated from a 1% TAE gel using a GENECLEAN IIIkit (BIO 101, Vista, Calif.) and sequenced as above.

Construction and Screening of a Human Heart cDNA Library

Poly A+ RNA was purified from human heart RNA (Clontech) using aFastTrack kit (Invitrogen, Corp.). DS-cDNA was synthesized from 8 μg ofpoly A+RNA according to Gubler and Hoffman (1983) with minormodifications. The resulting cDNA was ligated to BstXI adaptors(Invitrogen, Corp.) and the excess adaptors removed by exclusion columnchromatography. High molecular weight fractions of size-selected ds-cDNAwere ligated in pEXJ.BS, an Okayama and Berg expression vector modifiedfrom pcEXV (Miller and Germain, 1986) to contain BstXI and otheradditional restriction sites. A total of 4.45×10⁶ independent cloneswith a mean insert size of 2.5 kb were generated. The library was platedon agar plates (Ampicillin selection) in 127 primary pools; 50 poolswith 37,500 independent clones, 51 pools with 25,000 clones and 26 poolswith 50,000 clones. Glycerol stocks of the primary pools were combinedin 16 superpools of 8 and screened for hGALR2 by PCR using primers BB153and BB169, a forward primer from the second intracellular domain ofhGALR2 identified in the 5′ RACE fragment above. PCR was performed withthe Expand Long Template PCR System (Boehringer Mannheim) under thefollowing conditions: 1 min at 94° C., 4 min at 68° C. for 40 cycles,with a pre- and post-incubation of 5 min at 95° C. and 7 min at 68° C.,respectively. Primary pools from positive superpools were screened byPCR and then primary pool 169 was subdivided and screened by PCR. Onepositive subpool, 69-11, was subdivided into 20 pools of 1200 clonesplated on agar plates (ampicillin selection) Colonies were transferredto nitrocellulose membranes (Schleicher and Schuell, Keene, N.H.),denatured in 0.4 N NaOH, 1.5 M NaCl, renatured in 1M Tris, 1.5 M NaCl,and UV cross-linked. Filters were hybridized overnight at 40° C. in abuffer containing 50% formamide, 5×SSC, 7 mM TRIS, 1×Denhardt's solutionand 25 μg/ml salmon sperm DNA (Sigma Chemical Co.) and 10⁶ cpm/ml ofKS1567, an oligonucleotide probe from the 3′ end of the fifthtransmembrane domain of hGALR2, labeled with γ-³²P [ATP] (6000 Ci/mmol,NEN) using polynucleotide kinase (Boehringer Mannheim). Filters werewashed 2×15 minutes at room temperature in 2×SSC, 0.1% SDS, 2×15 minutesat 50° C. in 0.1×SSC, 0.1% SDS, and exposed to XAR X-ray film (Kodak)for 3 days. Colonies which appeared to hybridize were re-screened by PCRusing primers BB167 and BB170, a reverse primer from the COOH terminusof hGlR2 identified by the 3′ RACE fragment above. PCR was performedwith the Expand Long Template PCR System (Boehringer Mannheim) under thefollowing conditions: 1 min at 94° C., 2 min at 58° C., 2 min at 68° C.for 28 cycles, with a pre- and post-incubation of 5 min at 95° C. and 7min at 68° C. respectively. One positive colony, 69-11-5 was amplifiedovernight in 10 ml LB media and processed for plasmid purification usinga standard alkaline lysis miniprep procedure followed by a PEGprecipitation. To ensure that 69-11-5 was a single colony, it wasamplified for 3 hours in 3 ml of LB media and then 1 μl of a 1:100dilution was plated on an agar plate. Twenty colonies were screened byPCR using primers BB167 and BB170 using the same conditions as above,except that 25 cycles were used instead of 28. One positive singlecolony, 69-11-5-3, designated BO29, was amplified overnight in 10 ml ofTB media and processed for plasmid purification. Vector-anchored PCR wasperformed on BO29 using the Expand Long Template PCR System (BoehringerMannheim) to determine the orientation and size of the insert. BB173 andBB172, forward and reverse vector primers, respectively, were used withprimers BB169 and BB153. The conditions for PCR were 1 min at 94° C., 4min at 68° C. for 36 cycles, with a pre- and post-incubation of 5 min at95° C. and 7 min at 68° C. respectively. BO29 is preferably sequenced onboth strands using AmpliTaq DNA Polymerase, FS (Perkin Elmer) Thesequence is run on an ABI PRISM 377 DNA Sequencer and analyzed using theWisconsin Package (GCG, Genetics Computer Group, Madison, Wis).

To test the ability of 69-11-5 to confer galanin binding, COS-7 cellswere plated in slide chambers (Lab-Tek) in Dulbecco's modified Eaglemedium (DMEM) supplemented with 10% calf serum, 100 U/ml of penicillin,100 μg/ml streptomycin, 2 mM L-glutamine (DMEM-c) and grown at 37° C. ina humidified 5% CO₂ atmosphere for 24 hours before transfection. Cellswere transfected with 1 μg of miniprep DNA from 69-11-5 or vectorcontrol using a modification of the DEAE-dextran method (Warden andThorne, 1968). 48 hours after transfection, cells were rinsed withphosphate-buffered saline (PBS) then incubated with 1 nM ¹²⁵I-ratgalanin (NEN; specific activity ˜2200 Ci/mmol) and 2 nM ¹²⁵I-porcinegalanin (NEN; specific activity ˜2200 Ci/mmol) in 20 mM HEPES-NaOH, pH7.4, containing 1.26 mM CaCl₂, 0.81 mM MgSO₄, 0.44 mM KH₂PO₄, 5.4 mMKCl, 10 mM NaCl, 0.1% BSA, and 0.1% bacitracin for one hour at roomtemperature. After rinsing and fixation in 2.5% glutaraldehyde, slideswere rinsed in PBS, air-dried, and dipped in photoemulsion (Kodak,NTB-2). After a 4-day exposure, slides were developed in Kodak D19developer, fixed, and coverslipped (Aqua-Mount, Lerner Laboratories),then inspected for positive cells by brightfield microscopy (LeitzLaborlux, 25× magnification). To test the ability of the single cloneBO29 to confer galanin binding, BO29 or control. vector were transfectedinto COS-7 cells for testing of ¹²⁵I galanin as described above, withthe exception that after fixation, binding of ¹²⁵I galanin to cells onthe slide was detected using an ¹²⁵I probe (Mini-Instruments, Ltd.,Essex, England). The signal from BO29 transfected cells was comparedwith the signal from control vector transfected cells.

Primers and Probes Used

NS525: 5′CCCTACCTGAGCTACTACCGTCA 3′ (SEQ ID NO:15);

NS526: 5′ACCAAACCACACGCAGAGGATAAG 3′ (SEQ ID NO:16);

BB153: 5′-CCACGATGAGGATCATGCGTGTCACC-3′ (SEQ ID NO:17);

BB154: 5′-TAGGTCAGGCCGAGAACCAGCACAGG-3′ (SEQ ID NO:18);

NS563: 5′-CAGGTAGCTGAAGACGAAGGTGCA-3′ (SEQ ID NO:19);

BB166: 5′-CTGCACCTTCGTCTTCAGCTACCTG-3′ (SEQ ID NO:20);

BB167: 5′-CCTGTGCTGGTTCTCGGCCTGACCTA-3′ (SEQ ID NO:21);

BB169: 5′-TATCTGGCCATCCGCTACCCGCTGCA-3′ (SEQ ID NO:22);

KS1567: 5′-TTGCGCTACCTCTGGCGCGCCGTCGACCCGGTGGCCGCGGGCTCG-3′ (SEQ IDNO:23);

BB170: 5′-CCAACAATGACTCCAACTCTGTGAC-3′ (SEQ ID NO:24);

BB173: 5′-AGGCGCAGAACTGGTAGGTATGGAA-3′ (SEQ ID NO:25); and

BB172: 5′-AAGCTTCTAGAGATCCCTCGACCTC-3′ (SEQ ID NO:26).

Generation of an Intronless Human GALR2 Receptor

Human tissues may be screened by PCR, using primers that cross theintron, to identify cDNA sources that express the intronless form. Anintronless hGALR2 clone may be obtained using an approach similar tothat used to obtain an intronless rGALR2 clone (infra). Alternatively,one may use restriction enzymes to remove the intron and some adjacentcoding region from BO29, and then replace the removed coding region byinserting a restriction enzyme-digested PCR fragment amplified from atissue shown to express the intronless form of the receptor.

Human hippocampus and human hypothalamus were each shown to express theintronless form. A full-length, intronless human GALR2 PCR product wasamplified from human hippocampus, but was found to contain a singlepoint mutation downstream from the intron splice site. Therefore, anEcoRI/StyI restriction digest fragment, containing 11 bp of 5′ UT andthe first 557 bp of hGalR2 coding region, was ligated to a StyIrestriction digest fragment, containing bp 558-1164 of the coding regionand 182 bp of 3′ UT, which was isolated from the intron-containinghGALR2 clone (BO29). The ligation product, comprising the entireintronless form of the human GALR2 receptor, was subcloned into thevector pEXJ and designated BO39 (ATCC Accession No. 97851).

Northern Blots

Human brain multiple tissue northern blots (MTN blots II and III,Clontech, Palo Alto, Calif.) carrying mRNA purified from various humanbrain areas may be hybridized according to the manufacturers'specifications.

Rat multiple tissue northern blots including multiple brain tissue blots(rat MTN blot, Clontech, Palo Alto, Calif.) carrying mRNA purified fromvarious rat tissues also may be hybridized at high stringency accordingto the manufacturer's specifications.

RT-PCR Analyses of GALR2 mRNA

Tissues may be homogenized and total RNA extracted using the guanidineisothiocyanate/CsCl cushion method. RNA may then be treated with DNaseto remove any contaminating genomic DNA. cDNA may be prepared from totalRNA with random hexanucleotide primers using the reverse transcriptaseSuperscript II (BRL, Gaithersburg, Md.). First strand cDNA (about 250 ngof total RNA) may be amplified for example, in a 50 μL PCR reactionmixture (200 μM dNTPs final concentration) and 1 μM appropriate primers,using an appropriate thermal cycling program.

The PCR products may be run on a 1.5% agarose gel and transferred tocharged nylon membranes (Zetaprobe GT, BioRad), and analyzed as Southernblots. GALR2 primers will be screened for the absence ofcross-reactivity with the other galanin receptors. Filters may behybridized with radiolabeled probes and washed under high stringency.Labeled PCR products may be visualized on X-ray film. Similar PCR andSouthern blot analyses may be conducted with primers and probes, e.g.,1B15, directed to the housekeeping gene, glyceraldehyde phosphatedehydrogenase (Clontech, Palo Alto, Calif.), to normalize the amount ofcDNA used from the different tissues.

RT PCR of rat brain tissues was carried out using total or poly A⁺ RNA(1.5 μg or 0.5 μg, respectively) isolated from various rat brain regionsand converted to cDNA using Superscript II (BRL, Gaithersburg, Md.)reverse transcriptase with random priming. The cDNAs were used astemplates for PCR amplification of GALR2 using specific GALR2 primers.PCR products were separated on an agarose gel by electrophoresis andblotted to a charged nylon membrane.

RT-PCR of human tissues was carried out using cDNAs prepared from totalRNA as described above (using hippocampal, hypothalamic, heart, kidney,liver, lung and retinal RNAs purchased from Clontech, Palo Alto, Calif.)or using cDNAs purchased from Clontech (for cerebral cortex, adrenalgland, lymph node, small intestine, spleen and stomach tissues). Theprimers used were BB182 (reverse primer from hGALR2 stop codon) andBB183 (forward primer from hGALR2 start codon). The PCR products wereSouthern blotted and hybridized with a ³²P-labeled oligonucleotideprobe, BB247.

Primers and probe sequences:

BB182: 5′-TCGTAATAGAAGCTTGGCCACATCAACCGTCAGGATGCTG-3′ (SEQ ID NO: 33);

BB183: 5′-TATCGATAGGAATTCAGCGGCACCATGAACGTCTCGGGCT-3′ (SEQ ID NO: 34);and

BB247: 5′-ATCGTTTACGCGCTGGTCTCCAAGCACTTCCGCAAAGGCTT CCGCACGAT-3′ (SEQ IDNO: 35).

Isolation of the Intronless Rat GALR2

RT-PCR analysis of various rat brain regions (FIG. 5) was carried outusing primers representing N- and C-termini of rat GALR2 (supra). Theforward and reverse primers comprised nucleotides 1-23 and 1087-1110,respectively, of the intronless rat GALR2 sequence (SEQ. ID No. 7). ThePCR products were separated by agarose gel electrophoresis, blotted, andhybridized with an oligonucleotide probe designed to the predicted 5/6loop of GALR2 (nucleotides 651-695, SEQ. ID No. 7). This analysisindicated the presence of both intron-containing and intronless forms ofrat GALR2 in brain. In order to choose an appropriate tissue source fromwhich to isolate the intronless form, a similar PCR analysis on RNA froma variety of rat tissues was carried out. Based on the size of theproducts determined by agarose gel electrophoresis (data not shown), ratheart was chosen as a potential source of intronless GALR2 RNA. Toisolate the intronless GALR2, PCR primers similar to those used abovebut containing restriction enzyme sites to facilitate subcloning and aKozak consensus for translation initiation (KS-1550 and KS-1551, seebelow) were used to amplify rat GALR2 from rat heart RNA by PCR (afterconversion of the RNA to first strand cDNA by standard methods). A PCRproduct of the correct size was isolated from an agarose gel and thenreamplified using the same primers to increase yield. The products weredigested with the appropriate restriction enzymes to produce cohesiveends (EcoRI and Xba I), ligated into the expression vector EXJ.RH andtransformed into E.coli. The resulting colonies were transferred tonitrocellulose membranes and hybridized with an oligonucleotide probe tothe predicted 2/3 loop of rat GALR2 (nucleotides 259-303, SEQ. ID No.7). A single hybridizing colony was found by subsequent analysis tocontain the intronless rat GALR2 cDNA.

Primers used:

Forward primer, KS-1550: 5′-ACGGAATTCGACATGAATGGCTCCGGCA (SEQ. ID No.27)

Reverse Primer, KS-1551: 5′-GCTCTAGAGCCCCTTTGGTCCTTTAACAAGCCGG (SEQ. IDNo. 28)

Production of Recombinant Baculovirus

The coding region of GALR2 may be subcloned into pBlueBacIII intoexisting restriction sites, or sites engineered into sequences 5′ and 3′to the coding region of GALR2, for example, a 5′ BamHI site and a 3′EcoRI site. To generate baculovirus, 0.5 μg of viral DNA (BaculoGold)and 3 μg of GALR2 construct may be co-transfected into 2×10⁶ Spodopterafrugiperda insect Sf9 cells by the calcium phosphate co-precipitationmethod, as outlined in by Pharmingen (in “Baculovirus Expression VectorSystem: Procedures and Methods Manual”). The cells then are incubatedfor 5 days at 27° C.

The supernatant of the co-transfection plate may be collected bycentrifugation and the recombinant virus plaque purified. The procedureto infect cells with virus, to prepare stocks of virus and to titer thevirus stocks are as described in Pharmingen's manual.

Cell Culture

COS-7 cells are grown on 150 mm plates in DMEM with supplements(Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mMglutamine, 100 units/mL penicillin/100 μg/mL streptomycin) at 37° C., 5%CO₂. Stock plates of COS-7 cells are trypsinized and split 1:6 every 3-4days. Human embryonic kidney 293 cells are grown on 150 mm plates inDMEM with supplements (10% bovine calf serum, 4 mM glutamine, 100units/mL penicillin/100 μg/mL streptomycin) at 37° C., 5% CO₂. Stockplates of 293 cells are trypsinized and split 1:6 every 3-4 days. Mousefibroblast LM(tk−) cells are grown on 150 mm plates in D-MEM withsupplements (Dulbecco's Modified Eagle Medium with 10% bovine calfserum, 4 mM glutamine, 100 units/mL penicillin/100 μg/mL streptomycin)at 37° C., 5% CO₂. Stock plates of LM(tk−) cells are trypsinized andsplit 1:10 every 3-4 days. Chinese hamster ovary (CHO) cells were grownon 150 mm plates in HAM's F-12 medium with supplements (10% bovine calfserum, 4 mM L-glutamine and 100 units/mL penicillin/100 ug/mlstreptomycin) at 37° C., 5% CO2. Stock plates of CHO cells weretrypsinized and split 1:8 every 3-4 days.

LM(tk−) cells stably transfected with the GALR2 receptor may beroutinely converted from an adherent monolayer to a viable suspension.Adherent cells are harvested with trypsin at the point of confluence,resuspended in a minimal volume of complete DMEM for a cell count, andfurther diluted to a concentration of 10⁶ cells/mL in suspension media(10% bovine calf serum, 10% 10× Medium 199 (Gibco), 9 mM NaHCO₃, 25 mMglucose, 2 mM L-glutamine, 100 units/mL penicillin/100 μg/mLstreptomycin, and 0.05% methyl cellulose). Cell suspensions aremaintained in a shaking incubator at 37° C., 5% CO₂ for 24 hours.Membranes harvested from cells grown in this manner may be stored aslarge, uniform batches in liquid nitrogen. Alternatively, cells may bereturned to adherent cell culture in complete DMEM by distribution into96-well microtiter plates coated with poly-D-lysine (0.01 mg/mL)followed by incubation at 37° C. 5% CO₂ for 24 hours. Cells prepared inthis manner generally yield a robust and reliable response in cAMPradio-immunoassays as further described hereinbelow.

Mouse embryonic fibroblast NIH-3T3 cells are grown on 150 mm plates inDulbecco's Modified Eagle Medium (DMEM) with supplements (10% bovinecalf serum, 4 mM glutamine, 100 units/mL penicillin/100 μg/mLstreptomycin) at 37° C., 5% CO2. Stock plates of NIH-3T3 cells aretrypsinized and split 1:15 every 3-4 days.

Sf9 and Sf21 cells are grown in monolayers on 150 mm tissue culturedishes in TMN-FH media supplemented with 10% fetal calf serum, at 27°C., no CO₂. High Five insect cells are grown on 150 mm tissue culturedishes in Ex-Cell 400™ medium supplemented with L-Glutamine, also at 27°C., no CO₂.

Transfection

All receptor subtypes studied may be transiently transfected into COS-7cells by the DEAE-dextran method, using 1 μg of DNA /10⁶ cells (Cullen,1987). In addition, Schneider 2 Drosophila cells may be cotransfectedwith vectors containing the receptor gene, under control of a promoterwhich is active in insect cells, and a selectable resistance gene, eg.,the G418 resistant neomycin gene, for expression of the galaninreceptor.

Stable Transfection

The GALR2 receptor may be co-transfected with a G-418 resistant geneinto the human embryonic kidney 293 cell line by a calcium phosphatetransfection method (Cullen, 1987). GALR1 receptors were expressed incells using methods well-known in the art. Stably transfected cells areselected with G-418. GALR2 receptors may be similarly transfected intomouse fibroblast LM(tk−) cells, Chinese hamster ovary (CHO) cells andNIH-3T3 cells. Transfection of LM(tk−) cells with the plasmid K985 andsubsequent selection with G-418 resulted in the LM(tk−) cell lineL-rGALR2-8 (ATCC Accession No. CRL-12074), which stably expresses therat GALR2 receptor. A similar procedure was used to transfect LM(tk−)cells with plasmid K1045 (intronless rat GALR2 receptor construct)resulting in the LM(tk−) cell line L-rGALR4-I (ATCC Accession No.CRL-12223). In addition, this procedure was used to transfect CHO cellswith an intron-containing plasmid to create a stably expressing ratGALR2 CHO cell line, C-rGalR2-79 (ATCC Accession No. CRL-12262).Transfection of CHO cells with the (intronless) plasmid B039 andsubsequent selection with G-418 resulted in the cell line CHO-hGALR2-264(ATCC Accession No. CRL-), which stably expresses the human GALR2receptor.

Radioligand Binding Assays

Transfected cells from culture flasks were scraped into 5 ml ofTris-HCl, 5 mM EDTA, pH 7.5, and lysed by sonication. The cell lysateswere centrifuged at 1000 rpm for 5 min. at 4° C. and the supernatant wascentrifuged at 30,000×g for 20 min. at 4° C. The pellet was suspended inbinding buffer (50 mM Tris-HCl, 5 mM MgSO₄, 1 mM EDTA at pH 7.5supplemented with 0.1% BSA, 2 μg/ml aprotinin, 0.5 mg/ml leupeptin, and10 μg/ml phosphoramidon). Optimal membrane suspension dilutions, definedas the protein concentration required to bind less than 10% of the addedradioligand, were added to 96-well polpropylene microtiter platescontaining ¹²⁵I-labeled peptide, non-labeled peptides and binding bufferto a final volume of 250 μl. In equilibrium saturation binding assaysmembrane preparations were incubated in the presence of increasingconcentrations (0.1 nM to 4 nM) of [¹²⁵I]porcine galanin (specificactivity 2200 Ci/mmol) The binding affinities of the different galaninanalogs were determined in equilibrium competition binding assays, using0.1 nM [¹²⁵I]porcine galanin in the presence of twelve differentconcentrations of the displacing ligands. Binding reaction mixtures wereincubated for 1 hr at 30° C. and the reaction was stopped by filtrationthrough GF/B filters treated with 0.5% polyethyleneimine, using a cellharvester. Radioactivity was measured by scintillation counting and datawere analyzed by a computerized non-linear regression program.Non-specific binding was defined as the amount of radioactivityremaining after incubation of membrane protein in the presence of 100 nMof unlabeled porcine galanin. Protein concentration was measured by theBradford method using Bio-Rad Reagent, with bovine serum albumin as astandard.

Binding assays involving the rat GALR3 receptor are conducted at roomtemperature for 120 min. in binding buffer. Leupeptin, aprotonin andphosphoramidon are omitted from rat GALR3 assays while bacitracin isadded to 0.1%. Nonspecific binding is defined in the presence of 1 μMporcine galanin. Cells transiently or stably expressing GALR3 receptorsare produced using transfection. methods which are well-known in theart, examples of which are provided herein (supra). The rat GALR3receptor may be expressed using plasmid K1086, deposited on Oct. 8,1996, with the ATCC, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.Aunder the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure, and wasaccorded ATCC Accession No. 97747. Another plasmid expressing the ratGALR3 receptor is plasmid pEXJ-rGALR3t, deposited with the ATCC underthe Budapest Treaty on Dec. 17, 1996, and accorded ATCC Accession No.97826. The human GALR3 receptor may be expressed using plasmidpEXJ-hGALR3, also deposited with the ATCC under the Budapest Treaty onDec. 17, 1996, and accorded ATCC Accession No. 97827. Cells stablyexpressing the GALR3 receptors may be used in functional assays wellknown in the art, examples of which are provided herein (infra).

Functional Assays

Cyclic AMP (cAMP) Formation

The receptor-mediated inhibition of cyclic AMP (cAMP) formation may beassayed in LM(tk−), CHO or 293 cells expressing the rat GALR1 and GALR2receptors. Cells were plated in 96-well plates and incubated inDulbecco's phosphate buffered saline (PBS) supplemented with 10 mMHEPES, 5 mM theophylline, 2 μg/ml aprotinin, 0.5 mg/ml leupeptin, and 10μg/ml phosphoramidon for 20 min at 37° C., in 5% CO₂. Galanin or thetest compounds were added and incubated for an additional 10 min at 37°C. The medium was aspirated and the reaction was stopped by the additionof 100 mM HCl. The plates were stored at 4° C. for 15 min, and the cAMPcontent in the stopping solution was measured by radioimmunoassay.Radioactivity was quantified using a gamma counter equipped with datareduction software.

Functional assay experiments were also performed using stablytransfected cells seeded into 96-well microtiter plates and cultureduntil confluent. To reduce the potential for receptor desensitization,the serum component of the media was reduced to 1.5% for 4 to 16 hoursbefore the assay. Cells were washed in Hank's buffered saline, or HBS(150 mM NaCl, 20 mM HEPES, 1 mM CaCl₂, 5 mM KCl, 1 mM MgCl₂, and 10 mMglucose) supplemented with 0.1% bovine serum albumin plus 5 mMtheophylline and pre-equilibrated in the same solution for 20 min at 37°C. in 5% CO₂. Cells were then incubated 5 min with 10 μM forskolin andvarious concentrations of receptor-selective ligands. The assay wasterminated by the removal of HBS and acidification of the cells with 100mM HCl. Intracellular cAMP was extracted and quantified with a modifiedversion of a magnetic bead-based radioimmunoassay (Advanced Magnetics,Cambridge, Mass.). The final antigen/antibody complex was separated fromfree ¹²⁵I-cAMP by vacuum filtration through a PVDF filter in amicrotiter plate (Millipore, Bedford, Mass.). Filters were punched andcounted for 125I in a Packard gamma counter. Functional studies of therat GALR1 receptor in LMTK− cells were performed as previously describedabove except that leupeptin, aprotinin and phosphoramidon were omittedfrom the assay, and cells were stimulated with forskolin plus peptidesfor a period of 5 min.

Arachidonic Acid Release

CHO cells stably transfected with the rat GALR2 receptor were seededinto 96 well plates and grown for 3 days in HAM's F-12 with supplements.³H-arachidonic acid (specific activity=0.75 uCi/ml) was delivered as a100 ul aliquot to each well and samples were incubated at 37° C., 5% CO₂for 18 hours. The labeled cells were washed three times with 200 ulHAM's F-12. The wells were then filled with medium (200 uL) and theassay was initiated with the addition of peptides or buffer (22 uL).Cells were incubated for 30 min at 37° C., 5% CO2. Supernatants weretransferred to a microtiter plate and evaporated to dryness at 75° C. ina vacuum oven. Samples were then dissolved and resuspended in 25 uLdistilled water. Scintillant (300 uL) was added to each well and sampleswere counted for ³H in a Trilux plate reader. Data were analyzed usingnonlinear regression and statistical techniques available in theGraphPAD Prism package (San Diego, Calif.).

Intracellular Calcium Mobilization

The intracellular free calcium concentration may be measured bymicrospectroflourometry using the fluorescent indicator dye Fura-2/AM(Bush et al. 1991). Stably transfected cells are seeded onto a 35 mmculture dish containing a glass coverslip insert. Cells are washed withHBS and loaded with 100 μL of Fura-2/AM (10 μM) for 20 to 40 min. Afterwashing with HBS to remove the Fura-2/AM solution, cells areequilibrated in HBS for 10 to 20 min. Cells are then visualized underthe 40× objective of a Leitz Fluovert FS microscope and fluorescenceemission is determined at 510 nM with excitation wavelengths alternatingbetween 340 nM and 380 nM. Raw fluorescence data are converted tocalcium concentrations using standard calcium concentration curves andsoftware analysis techniques.

Phosphoinositide Metabolism

LM(tk−) cells stably expressing the rat GALR2 receptor cDNA were platedin 96-well plates and grown to confluence. The day before the assay thegrowth medium was changed to 100 μl of medium containing 1% serum and0.5 μCi [³H]myo-inositol, and the plates were incubated overnight in aCO₂ incubator (5% CO₂ at 37° C.). Alternatively, arachidonic acidrelease may be measured if [³H]arachidonic acid is substituted for the[³H]myo-inositol. Immediately before the assay, the medium was removedand replaced by 200 μL of PBS containing 10 mM LiCl, and the cells wereequilibrated with the new medium for 20 min. During this interval cellswere also equilibrated with the antagonist, added as a 10 μL aliquot ofa 20-fold concentrated solution in PBS. The [³H]inositol-phosphatesaccumulation from inositol phospholipid metabolism was started by adding10 μL of a solution containing the agonist. To the first well 10 μL wereadded to measure basal accumulation, and 11 different concentrations ofagonist were assayed in the following 11 wells of each plate row. Allassays were performed in duplicate by repeating the same additions intwo consecutive plate rows. The plates were incubated in a CO₂ incubatorfor 1 hr. The reaction was terminated by adding 15 μl of 50% v/vtrichloroacetic acid (TCA), followed by a 40 min. incubation at 4° C.After neutralizing TCA with 40 μl of 1M Tris, the content of the wellswas transferred to a Multiscreen HV filter plate (Millipore) containingDowex AG1-X8 (200-400 mesh, formate form). The filter plates wereprepared adding 200 μL of Dowex AG1-X8 suspension (50% v/v, water:resin) to each well. The filter plates were placed on a vacuum manifoldto wash or elute the resin bed. Each well was washed 2 times with 200 μLof water, followed by 2×200 μL of 5 mM sodium tetraborate/60 mM ammoniumformate. The [³H]IPs were eluted into empty 96-well plates with 200 μlof 1.2 M ammonium formate/0.1 formic acid. The content of the wells wasadded to 3 mls of scintillation cocktail, and the radioactivity wasdetermined by liquid scintillation counting.

GTPγS Functional Assay

Membranes from cells transfected with the rat GalR2 receptor weresuspended in assay buffer (50 mM Tris, 100 mM NaCl, 5 mM MgCl₂, pH 7.4)supplemented with 0.1% BSA, 0.1% bacitracin and 10 μM GDP. Membraneswere incubated on ice for 20 minutes, transferred to a 96-well Milliporemicrotiter GF/C filter plate and mixed with GTPγ³⁵S (250,000 cpm/sample,specific activity ˜1000 Ci/mmol) plus or minus GTPγS (finalconcentration=100 μM). Final membrane protein concentration=90 μg/mL.Samples were incubated in the presence or absence of porcine galanin(final concentration=1 μM) for 30 min. at room temperature, thenfiltered on a Millipore vacuum manifold and washed three times with coldassay buffer. Samples collected in the filter plate were treated withscintillant and counted for ³⁵S in a Trilux (Wallac) liquidscintillation counter.

MAP Kinase Assay

CHO cells expressing either the rat GALR2 receptor or the human GALR2receptor were plated at ˜50% confluence into well-plates and grown inculture with fetal bovine serum reduced to 0.5% for 2 days. Fresh mediumwas exchanged for old and then aspirated after 2 hrs. The assay wasinitiated by adding fresh medium plus or minus 1 uM human galanin for 10minutes. Cells were washed with phosphate buffered saline and lysed in100 uL Laemmli sample buffer (Bio-Rad) containing 5% β-mercaptoethanol.Cell lysate was scraped off the plate, transferred to a microfuge tubeon ice, and sonicated for 10-15 seconds. Samples were heated at 100° C.for 3-5 minutes. Insoluble material was collected by centrifugation at10,000×g for 5 min. Supernatant (20 μL/lane) was loaded onto a 4-20%polyacrylamide Bio-Rad Ready-Gel (10×10 cm) together with a phospho-MAPkinase protein standard (New England BioLabs) and molecular weightmarkers. Proteins were fractionated by SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) and transferred by electrophoresisto a PVDF membrane (Bio-Rad). The membrane was blocked with 5% nonfatdry milk. Phosphorylated MAP kinase was labeled with a rabbitanti-phospho-MAP kinase antibody (New England New England BioLabs) andHRP-conjugated goat anti-rabbit IgG according to standard western blotprocedure. The phospho-MAP kinase complex was detected bychemiluminescence using Lumiglow reagent (New England BioLabs) and KodakX-Omat film, with exposure times ranging from 10 to 120 seconds.

Functional assays using GALR3 receptors are performed similarly.

It is to be understood that the cell lines described herein are merelyillustrative of the methods used to evaluate the binding and function ofthe galanin receptors of the present invention, and that other suitablecells may be used in the assays described herein.

Functional Responses in Oocytes Expressing GalR2

Female Xenopus laevis (Xenopus-1, Ann Arbor, Mich.) were anesthetized in0.2% tricain (3-aminobenzoic acid ethyl ester, Sigma Chemical Corp.) anda portion of ovary was removed using aseptic technique (Quick andLester, 1994). Oocytes were defolliculated using 2 mg/ml collagenase(Worthington Biochemical Corp., Freehold, N.J.) in a solution containing87.5 mM NaCl, 2 mM KCl, 2 mM MgCl₂ and 5 mM HEPES, pH 7.5. Oocytes wereinjected (Nanoject, Drummond Scientific, Broomall, Pa.) with 50 nL ofrat GALR2 mRNA or other mRNA for use as a negative control. RNA wasprepared by linearization of the plasmid (pBluescript) containing theentire coding region of the GALR2 cDNA, followed by in vitrotranscription using the T7 polymerase (“MessageMachine”, Ambion).Alternatively, mRNA may be translated from a template generated by PCR,incorporating a T7 promoter. Oocytes were incubated at 16° on a rotatingplatform for 3-8 days post-injection. Dual electrode voltage clamp(“GeneClamp”, Axon Instruments Inc., Foster City, Calif.) was performedusing 3 M KCl-filled glass microelectrodes having resistances of 1-3Mohms. Unless otherwise specified, oocytes were clamped at a holdingpotential of −80 mV. During recordings, oocytes are bathed incontinuously flowing (2-5 ml/min) medium containing 96 mM NaCl, 2 mMKCl, 2 mM CaCl₂, 2 mM MgCl₂, 5 mM HEPES, pH 7.5 (ND96). Drugs areapplied by switching from a series of gravity fed perfusion lines.

Aequorin Assays of Calcium Mobilization in Oocytes

Aequorin assays are performed by injecting oocytes with 74 ng ofrecombinant aequorin protein (Molecular Probes, Inc.) two or three daysafter the initial injection with (for example) rGALR2 mRNA. One daylater the oocytes are incubated at room temperature, in the dark, in 5μM coelenterazine (Molecular Probes) for 4 hours. Oocytes are placedindividually in the well of a microtiter dish and stimulated withporcine galanin at a final concentration of 100 nM. Luminescence ismeasured over a two minute interval using a Dynex MLX luminometer.Values are reported in relative light units and represent a summation ofthe luminescence over that time period.

The human GALR2 receptor and GALR3 receptors may be studied functionallyusing similar methods.

For functional studies of GALR3 in Xenopus oocytes, oocytes wereprepared as above, except that the oocytes were defolliculated using 3mg/ml collagenase. Oocytes were injected (Nanoject, Drummond Scientific,Broomall, Pa.) 24 h later with 50-70 nL of individual mRNAs or mRNAmixtures (see below).

Genes encoding G-protein inwardly rectifying K+ channels 1 and 4 (GIRK1and GIRK4) were obtained by PCR using the published sequences (Kubo etal., 1993; Dascal et al., 1993; Krapivinsky et al., 1995) to deriveappropriate 5′ and 3′ primers. Human heart cDNA was used as templatetogether with the primers 5′-CGCGGATCCATTATGTCTGCACTCCGAAGGAAATTTG-3′(SEQ ID NO: 36) and 5′-CGCGAATTCTTATGTGAAGCGATCAGAGTTCATTTTTC-3′ (SEQ IDNO: 37) for GIRK1 and 5′-GCGGGATCCGCTATGGCTGGTGATTCTAGGAATG-3′ (SEQ IDNO: 38) and 5′-CCGGAATTCCCCTCACACCGAGCCCCTGG-3′ (SEQ ID NO: 39) forGIRK4. In each primer pair, the upstream primer contained a BamHI siteand the downstream primer contained an EcoRI site to facilitate cloningof the PCR product into pcDNAl-Amp (Invitrogen). mRNAs were transcribedusing the T7 polymerase (“Message Machine”, Ambion). Each oocytereceived 2 ng each of GIRK1 and GIRK4 mRNA in combination with 25 ng ofGalR2 mRNA. After injection of mRNAs, oocytes were incubated at 17° for3-8 days.

Dual electrode voltage clamp (“GeneClamp”, Axon Instruments Inc., FosterCity, Calif.) was performed using 3 M KCl-filled glass microelectrodeshaving resistances of 1-3 Mohms. Unless otherwise specified, oocyteswere voltage clamped at a holding potential of −80 mV. Duringrecordings, oocytes were bathed in continuously flowing (1-3 mL/min)medium containing 96 mM NaCl, 2 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, and 5 mMHEPES, pH 7.5 (ND96), or, in the case of oocytes expressing GIRKs 1 and4, elevated K⁺ containing 48 mM KCl, 49 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂,and 5 mM HEPES, pH 7.5 (1/2 hK). Drugs were applied either by localperfusion from a 10 μL glass capillary tube fixed at a distance of 0.5mm from the oocyte, or for calculation of steady-state EC₅₀s, byswitching from a series of gravity fed perfusion lines. Experiments werecarried out at room temperature. All values are expressed asmean±standard error of the mean.

Galanin Receptor Autoradiography

Male Sprague-Dawley rats (Charles River, Wilmington, Mass.) wereeuthanized using CO₂, decapitated, and their brains immediately removedand frozen on dry ice. Tissue sections were cut at 20 μm using acryostat and thaw mounted onto gelatin coated slides. Tissues werepreincubated in two 10 minute changes of 50 mM Tris-HCl buffer pH 7.4,containing 5 mM MgSO₄ and 2 mM EGTA (Sigma). The radioligand binding wascarried out in the same buffer, which also contained 0.1% bovine serumalbumin, 0.02% aprotinin, 0.031% leupeptin, 0.1% phosphoramidate(Boehringer Mannheim), and 0.1 nM [¹²⁵I]porcine galanin (specificactivity 2200 Ci/mmol, NEN) for 1 hour at 22° C. Nonspecific binding wasdetermined in the presence of 5 μM porcine galanin (Bachem). As[D-Trp²]galanin₍₁₋₂₉₎ was shown to be selective for the cloned GAlR2receptor (infra), a 60 nM concentration of this peptide was used todisplace [¹²⁵I]galanin binding from the rat brain tissue sections. Theuse of this concentration was based on the binding data, which showedthe affinity of [D-Trp²]galanin₍₁₋₂₉₎ to be 6 nM at the GALR2 receptor,and 3 μM at the GALR1 receptor. In general, a 10× concentration of theblocking ligand is sufficient to remove 100% of the targeted receptor,while leaving the GALR1 receptor unaffected. After incubation, tissueswere dipped twice in ice-cold Tris-HCl buffer (40° C.), followed by a 5minute wash in ice-cold Tris-HCl buffer (4° C.), then dipped twice inice-cold deionized water to remove the salts. Sections were placed inX-ray cassettes and apposed to Dupont Cronex MRF 34 Film for 5 days.Films were developed using a Kodak M35A Processor.

Tissue Preparation for Neuroanatomical Studies

Male Sprague-Dawley Rats (Charles River) are decapitated and the brainsrapidly removed and frozen in isopentane. Coronal sections are cut at 11μm on a cryostat and thaw-mounted onto poly-L-lysine coated slides andstored at −80° C. until use. Prior to hybridization, tissues are fixedin 4% paraformaldehyde, treated with 5 mM dithiothreitol, acetylated in0.1 M triethanolamine containing 0.25% acetic anhydride, delipidatedwith chloroform, and dehydrated in graded ethanols.

Probes

Oligonucleotide probes employed to characterize the distribution of therat GALR2 receptor mRNA may be synthesized, for example, on a MilliporeExpedite 8909 Nucleic Acid Synthesis System. The probes are thenlyophilized, reconstituted in sterile water, and purified on a 12%polyacrylamide denaturing gel. The purified probes are againreconstituted to a concentration of 100 ng/μL, and stored at −20° C.Probe sequences may include DNA or RNA which is complementary to themRNA which encodes the GALR2 receptor.

Localization of GALR2 mRNA: In situ Hybridization Animals

Timed-pregnant female Sprague-Dawley rats were puchased from CharlesRiver. The day of birth for each litter was designated as postnatal day0 (P0). Brains were removed from pups on P0, P3, P5, P8, P10, P15, P20,and P25. The brains from the mothers were also removed and used as theadult comparison. All brains were sectioned in the coronal plane at 11μm and the sections thaw-mounted on to poly-1-lysine coated microscopeslides. The sections were then used for in situ hybridizationhistochemistry as described below.

Tissue Preparation

Prior to hybridization, tissues were fixed in 4% paraformaldehyde,treated with 5 mM dithiothreitol, acetylated in 0.1 M triethanolaminecontaining 0.25% acetic anhydride, delipidated with chloroform, anddehydrated in graded ethanols. The sections were prehybridized for onehour at 40° C. in hybridization buffer, which consisted of 50%formamide, 4×sodium citrate buffer (1×SSC=0.15 M NaCl and 0.015 M sodiumcitrate), 1×Denhardt's solution (0.2% polyvinylpyrrolidine, 0.2% Ficoll,0.2% bovine serum albumin), 50 mM dithiothreitol, 0.5 mg/ml salmon spermDNA, 0.5 mg/ml yeast tRNA, and 10% dextran sulfate.

In Situ Hybridization

32 mer oligonucleotide probes complementary to nucleotides 261-292 ofthe GALR2 mRNA were synthesized, purified, and 3′-end labeled with³⁵S-dATP (1200 Ci/mmol, New England Nuclear, Boston, Mass.) to aspecific activity of 10⁹ dpm/μg using terminal deoxynucleotidyltransferase (Boehringer Mannheim; Indianapolis, Ind.). The radiolabeledprobes were purified on Biospin 6 chromatography columns (Bio-Rad;Richmond, Calif.), and diluted in the hybridization buffer describedabove to a concentration of 1.5×10⁴ cpm/μl. One hundred μl of theradiolabeled probe was applied to each section, which was then coveredwith a Parafilm coverslip. Hybridization was carried out overnight inhumid chambers at 40 to 55° C. The following day the sections werewashed in two changes of 2×SSC for one hour at room temperature, in2×SSC for 30 min at 50-60° C. and finally in 0.1×SSC for 30 min at roomtemperature. Tissues were dehydrated in graded ethanols and apposed toKodak XAR-5 film for 2 weeks at −-20° C., then dipped in Kodak NTB3autoradiography emulsion diluted 1:1 with 0.2% glycerol water. Afterexposure at 40° C. for 4 weeks, the slides were developed in Kodak D-19developer, fixed, and counterstained with hematoxylin and eosin.

Localization of GALR2 mRNA: Ribonuclease Protection Assay (RPA)

Development of Probes

A cDNA fragment encoding a 467 BP fragment of the rGALR2 was subclonedinto a pBluescript plasmid vector. This construct was linearized withXba I or Sal I. T3 and T7 RNA polymerases were used to synthesize thesense and antisense strands of RNA respectively. Full-length RNAtranscripts were obtained using a full-length cDNA construct in the samevector.

A probe coding for rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH)gene, a constitutively expressed protein, was used concurrently. GAPDHis expressed at a relatively constant level in most tissue and itsdetection was used to compare expression levels of the rGalR2 gene indifferent tissues.

RNA Extraction

RNA was isolated from rat peripheral tissue as well as regions of theCNS using a LiCl precipitation protocol (Cathala et al.,. 1983). Tissuewas homogenized in 5M guanidine isothiocyanate, 50 mM TRIS, 10 mM EDTA,using 7 ml of lysis buffer/gram tissue. 4M LiCl were added (7 ml/mlhomogenate) and the mixture were stored at 40° C. for 24-48 hours.Homogenates were centrifuged and the pellets were resuspended in 3MLiCl, and centrifuged again. The pellets were resuspended in 0.1% sodiumdodecyl sulfate (SDS), extracted in phenol:chloroform:isoamyl alcohol(24:24:1) and the RNA ethanol precipitated. Yield and relative puritywere assessed by measuring absorbance A₂₆₀/A₂₈₀.

Synthesis of Probes

rGALR2 and GAPDH cDNA sequences preceded by phage polymerase promotersequences were used to synthesize radiolabeled riboprobes. Conditionsfor the synthesis of riboprobes were: 1-2 μl linearized template (1μg/μl), 1 μl of ATP, GTP, UTP (10 mM each), 2 μl dithiothreitol (0.1 M),20 units RNAsin RNAse inhibitor, 1-2 μl (15-20 units/μl) RNA polymerase,4 μl transcription buffer (Promega Corp.), and 5 μl α³²P-CTP (specificactivity 800 Ci/mmol). 0.1 mM CTP (0.02-1.0 μl) were added to thereactions, and the volume were adjusted to 20 μl with DEPC-treatedwater. Labeling reactions were incubated at 38° C. for 90 min, afterwhich 2 units of RQ1 RNAse-free DNAse (Promega Corp.) were added todigest the template. The riboprobes were separated from unincorporatednucleotide by a spun G-50 column (Select D G-50(RF); 5 Prime-3 Prime,Inc.). TCA precipitation and liquid scintillation spectrometry were usedto measure the amount of label incorporated into the probe. A fractionof all riboprobes synthesized were size-fractionated on 0.4 mm thick 5%acrylamide sequencing gels and autoradiographed to confirm that theprobes synthesized were full-length and not degraded.

Solution Hybridization/ribonuclease Protection Assay

For solution hybridization 2-15 μg of total RNA isolated from tissueswere used. Sense RNA synthesized using the full-length coding sequenceof the rGalR2 was used to characterize specific hybridization. Negativecontrols consisted of 30 μg transfer RNA (tRNA) or no tissue blanks. Allsamples were placed in 1.5-ml microfuge tubes and vacuum dried.Hybridization buffer (40 μl of 400 mM NaCl, 20 mM Tris, pH 6.4, 2 mMEDTA, in 80% formamide) containing 0.25-1.0×10⁶ counts of each probewere added to each tube. Samples were heated at 90° C. for 15 min, afterwhich the temperature were lowered to 45° C. for hybridization.

After hybridization for 14-18 hr, the RNA/probe mixtures were digestedwith RNAse A (Sigma) and RNAse T1 (Bethesda Research Labs). A mixture of2.0 μg RNAse A and 1000 units of RNAse T1 in a buffer containing 330 mMNaCl, 10 mM Tris (pH 8.0) and 5 mM EDTA (400 μl) was added to eachsample and incubated for 90 min at room temperature. After digestionwith RNAses, 20 μl of 10% SDS and 50 μg proteinase K were added to eachtube and incubated at 37° C. for 15 min. Samples were then extractedwith phenol/chloroform:isoamyl alcohol and precipitated in 2 volumes ofethanol for 1 hr at −70° C. tRNA was added to each tube (30 mg) as acarrier to facilitate precipitation. Following precipitation, sampleswere centrifuged, washed with cold 70% ethanol, and vacuum dried.Samples were dissolved in formamide loading buffer and size-fractionatedon a urea/acrylamide sequencing gel (7.6 M urea, 6% acrylamide inTris-borate-EDTA). Gels were dried and apposed to Kodak XAR-5 x-rayfilm.

In vivo Methods

The effects of galanin, galanin derivatives, and related peptides andcompounds were evaluated by intracerebroventricular (i.c.v.) injectionof the peptide or compound followed by measurement of food intake in theanimal. Measurement of food intake was performed for 3 hours afterinjection, but other protocols may also be used. Saline was injected asa control, but it is understood that other vehicles may be required ascontrols for some peptides and compounds. In order to determine whethera compound is a GALR2 antagonist, food intake in rats may be stimulatedby administration of (for example) the GALR2-selective peptide agonist[D-Trp₂]-galanin₍₁₋₂₉₎ through an intracerebroventricular (i.c.v.)cannula. A preferred anatomic location for injection is thehypothalamus, in particular, the paraventricular nucleus. Methods ofcannulation and food intake measurements are well-known in the art, asare i.c.v. modes of administration (Kyrkouli et al., 1990, Ogren et al.,1992). To determine whether a compound reduces [D-Trp₂]-galanin₍₁₋₂₉₎stimulated food intake, the compound may be administered eithersimultaneously with the peptide, or separately, either through cannula,or by subcutaneous, intramuscular, or intraperitoneal injection, or morepreferably, orally.

Materials

Cell culture media and supplements are from Specialty Media (Lavallette,N.J.). Cell culture plates (150 mm and 96-well microtiter) are fromCorning (Corning, N.Y.). Sf9, Sf21, and High Five insect cells, as wellas the baculovirus transfer plasmid, pBlueBacIII™, are purchased fromInvitrogen (San Diego, Calif.). TMN-FH insect medium complemented with10% fetal calf serum, and the baculovirus DNA, BaculoGold™, is obtainedfrom Pharmingen (San Diego, Calif..). Ex-Cell 400™ medium withL-Glutamine is purchased from JRH Scientific. Polypropylene 96-wellmicrotiter plates are from Co-star (Cambridge, Mass.). All radioligandsare from New England Nuclear (Boston, Mass.).

Galanin and related peptide analogs were either from Bachem California(Torrance, Calif.), Peninsula (Belmont, Calif.) ; or were synthesized bycustom order from Chiron Mimotopes Peptide Systems (San Diego, Calif.).

Bio-Rad Reagent was from Bio-Rad (Hercules, Calif.). Bovine serumalbumin (ultra-fat free, A-7511) was from Sigma (St. Louis. Mo.). Allother materials were reagent grade.

Experimental Results

Isolation of a GALR2 cDNA from Rat Hypothalamus

In order to clone additional members of the galanin receptor family, anexpression cloning strategy based on the potential presence of multiplegalanin receptors in hypothalamus was designed. Although recent evidenceindicated that GALR1 receptor mRNA was present in rat hypothalamus(Gustafson et al., 1996; Parker et al., 1995), not all aspects of thecloned GALR1 pharmacological profile match that observed forgalanin-mediated feeding (Crawley et al., 1993). These results suggestedthat the regulation of galanin-induced feeding may not be explained bythe presence of only GALR1 in the rat hypothalamus.

A randomly-primed cDNA expression library was constructed from rathypothalamus and screened by radioligand binding/photoemulsion detectionusing [¹²⁵I]-porcine galanin. The library consisted of 584 poolscontaining about 5,000 primary clones/pool for a total of about 3million clones with an average insert size of 2.2 kb. Pools positive forrat GALR1 (about 110) were eliminated from the screen. Remaining poolswere screened for radioligand binding using 1 nM [¹²⁵I]-porcine galanin;slides were inspected for positive cells by direct microscopicexamination. One positive pool (J126) was subdivided into 96 pools ofabout 90 clones each and rescreened for galanin binding. Preliminarypharmacology carried out on the positive subpool J126-10 indicated thatthe [¹²⁵I]-porcine galanin binding was not sensitive to inhibition bygalanin 3-29. 400 individual colonies of a positive pool (J26-10) werethen screened to find two single purified cDNA clones. J126-10-334 waschosen for further analysis and designated K985. PCR analysis usingthree independent GALR1 primer sets (see Methods; data not shown)confirmed that the newly isolated cDNA was distinct from GALR1 and thusencoded a new galanin receptor subtype, termed GALR2.

The isolated clone K985 carries a 3.8 kb insert. Sequence analysis ofthis cDNA revealed a complete coding region for a novel receptor proteinwhich we term GALR2 (see FIGS. 1 and 2). Searches of GenEMBL databasesindicated that the sequence was novel, and that the most similarsequence was that of the galanin receptor GALR1, followed by other Gprotein-coupled receptors (GPCR). The nucleotide and deduced amino acidsequences are shown in FIGS. 1 and 2, respectively. The nucleotidesequence of the coding region is ˜56% identical to rat GALR1 and ˜54%identical to human GALR1 and encodes a 372 amino acid protein with 38%and 40% amino acid identity to rat and human GALR1, respectively.Hydropathy plots of the predicted amino acid sequence reveal sevenhydrophobic regions that may represent transmembrane domains (TMs, datanot shown), typical of the G protein-coupled receptor superfamily. Inthe putative TM domains, GALR2 exhibits 48-49% amino acid identity withrat and human GALR1. Like most GPCRs, the GALR2 receptor containsconsensus sequences for N-linked glycosylation in the N-terminus(positions 2 and 11) as well as the predicted extracellular loop betweenTMs IV and V. The GALR2 receptor contains two highly conserved cysteineresidues in the first two extracellular loops that are believed to forma disulfide bond stabilizing the functional protein structure (Probst etal., 1992). GALR2 shows five potential phosphorylation sites for proteinkinase C in positions 138, 210, 227, 319, and 364, and two cAMP- andcGMP-dependent protein kinase phosphorylation sites in positions 232 and316. It should be noted that six out of the seven potentialphosphorylation sites are located in predicted intracellular domains,and therefore could play a role in regulating functional characteristicsof the GALR2 receptor (Probst et al., 1992).

Within the GALR2 cDNA K985 (J126-10-334) isolated from the rathypothalamus library, the coding region of GALR2 is interrupted by anintron of ˜1 kb (FIGS. 3A, 3B, and 3C). A cDNA containing an intron maybe produced by the action of reverse transcriptase on an incompletelyspliced form of messenger RNA. The heterologous expression of thecomplete protein product is not necessarily impeded by the presence ofthe intron in the coding region, because the intron can typically bespliced out prior to translation by the host cell machinery. In the caseof the GALR2 cDNA, the location of the intron combined with clearconsensus sequences for 5′ and 3′ splice junctions (FIGS. 3A and 3B)confirm that the intervening sequence represents an intron. As shown inFIG. 3C, splicing of the intron at the indicated sites recreates an openreading frame within a highly conserved region of the GPCR family, atthe end of TMIII (LDR/Y). It is of interest to note that several GPCRshave previously been reported to contain introns at this location,including the human dopamine D3, D4, and D5 receptors, the rat substanceP receptor, and the human substance K receptor (Probst et al., 1992). Inparticular, the rat 5-HT₇ receptor (Shen et al., 1993) contains anintron in exactly the same location as is now reported for GALR2, withinthe AG/G codon for the highly conserved amino acid arginine at the endof TMIII (FIG. 3C).

To explore the possibility that incompletely or alternately splicedforms GALR2 mRNAs are present in the rat brain, RT-PCR using GALR2 PCRprimers that are located in the coding region but that span the locationof the intron was carried out. The sequences of the PCR primers are:

KS-1515 (Forward primer): 5′-CAAGGCTGTTCATTTCCTCATCTTTC (loop betweenTMs II and III) (SEQ.ID No. 12).

KS-1499 (Reverse primer): 5′-TTGGAGACCAGAGCGTAAACGATGG (end ofTMVII)(SEQ.ID No. 13).

The PCR products were separated by gel electrophoresis, blotted, andhybridized with a radiolabeled oligonucleotide probe representing thepredicted loop between TMs V and VI. The sequence of the oligonucleotideis:

KS-1540: 5′-AGTCGACCCGGTGACTGCAGGCTCAGGTTCCCAGCGCGCCAAACG (SEQ. ID No.14).

RT-PCR analysis of GALR2 mRNA from various rat brain regions asdescribed above indicates the existence of PCR products that mayrepresent both the intronless (spliced) and intron-containing(incompletely spliced) forms of GALR2 (FIG. 5). In addition, PCRproducts intermediate in size between intronless and intron-containingproducts that hybridize at high stringency with the GALR2oligonucleotide probe KS-1540 are present and may represent additionalvariations in the GALR2 mRNA. One mechanism that could generate suchvariations is alternative splicing. These results suggest thatintronless transcripts exist in native tissue. A full-length intronlesscDNA encoding the rat GALR2 receptor has been amplified and subclonedfrom rat heart RNA, which when transiently or stably transfected intocells binds galanin with high affinity.

Northern Blot Analysis of GALR2 mRNA

To define the size and distribution of the mRNA encoding GALR2 Northernblot analysis of poly A⁺ RNA from various rat tissues and brain regionswas carried out. A ˜1.2 kb fragment of rat GALR2 containing the entirecoding region but not containing the intron (FIG. 1) was radiolabeled byrandom priming and used as a hybridization probe. Northern blotscontaining rat poly A⁺ RNA were hybridized at high stringency andapposed to film. A single transcript of ˜1.8-2.0 kb is detected after a4 day exposure of the autoradiogram at −80° C. using Kodak Biomax MSfilm with one Biomax MS intensifying screen. Within the brain, thehighest levels of GALR2 mRNA appear in hypothalamus (FIG. 6A). Amongvarious rat tissues, the GALR2 transcript is widely but unevenlydistributed: GALR2 mRNA is observed in brain, lung, heart, spleen, andkidney, with lighter bands in skeletal muscle, liver, and testis (FIG.7A). Both Northern blots were reprobed with 1B15 to confirm that similaramounts of mRNA were present in each lane (FIGS. 6B and 7B).

Pharmacological Characterization of GALR2

The pharmacology of GALR2 was studied in COS-7 cells transientlytransfected with the GALR2 cDNA, K985. Membrane preparations of Cos-7cells transfected with K985 displayed specific binding to [¹²⁵I]porcinegalanin. Scatchard analysis of equilibrium saturation binding datayielded a K_(d)=150 pM with a B_(max)=250 fmol/mg protein. Thepharmacological properties of the protein encoded by the GALR2 cDNA wereprobed by measuring the binding affinities of a series of galaninanologs, and compared to those of the rat GALR1 receptor expressed inthe same host cell line. As shown in Table 1, both GALR1 and GALR2receptors showed a high affinity for galanin₍₁₋₂₉₎, the physiologicalligand of these receptors. Both receptors also displayed high affinityfor the truncated analogs galanin₍₁₋₁₆₎ and galanin₍₁₋₁₅₎. Furthermore,the binding of [¹²⁵I]porcine galanin to either GALR1 or GALR2 atconcentrations up to 100 μM was not displaced by porcine galanin₍₃₋₂₉₎.However, the GALR2 receptor has 540- and 4200-fold higher affinity for[D-Trp²]porcine galanin₍ 1-29), and [D-Trp²]galanin₍₁₋₁₆₎, respectively,than the GALR1 subtype. Also, [Ala⁵]galanin₍₁₋₆₎, and[Phe²]galanin₍₁₋₁₅₎ were moderately selective, with 15- and 17-foldgreater affinities for the GALR2 receptor than for the GALR1 receptorsubtype, respectively. [Ala⁹]galanin₍₁₋₆₎ was the only analog that wasfound to have the opposite selectivity, with 70-fold higher affinity forthe GALR1 receptor than for the GALR2 receptor. Interestingly, these tworeceptor subtypes showed no significant differences in their bindingaffinities for the chimeric galanin antagonists, galantide, C7, M32,M35, and M40.

In LM(tk−) cells stably expressing the rat GALR2 receptor cDNA, porcinegalanin₍₁₋₂₉₎ was found to inhibit the formation of cyclic AMP inducedby 10 μM forskolin. The effects of galanin were dose dependent with anEC₅₀=0.26±0.13 nM (n=3) (FIG. 9A). In the same cell line porcinegalanin₍₁₋₂₉₎ stimulated the formation of [³H]inositol phosphates, withan EC₅₀=112 nM (FIG. 9B). The phosphoinositide response mediated by therat. GALR2 receptor suggests that this receptor can also couple to theintracellular calcium mobilization and diacylglycerol pathway. However,the 400-fold lower EC₅₀ of porcine galanin₍₁₋₂₉₎ suggests that the GALR2receptor couples with low efficiency to this signaling pathway. Insupport of this notion stands the observation that porcine galanin₍1-29) had no effect on intracellular calcium levels in COS-7 cellstransfected with the cDNA encoding the rat GALR2 receptor. Thus, thedata presented herein suggest that the GALR2 receptor couplespreferentially to G_(ialpha), since the stimulation of phosphoinositidemetabolism and intracllular calcium mobilization are a hallmark orreceptors to the G_(qa) family of G-proteins. Furthermore, the datapresented herein also indicate that the inhibition of cAMP formation, aswell as the stimulation of phosphoinositide metabolism, can be used asfunctional assays to measure receptor activity in heterologous cellsystems expressing the rat GALR2 receptor. However, separate experimentsusing mouse fibroblast 293 cell lines stably expressing rat GALR2receptors did not provide evidence for galanin-dependent inhibition offorskolin-stimulated cAMP.

In subsequent experiments, the inhibitory effect of rat GALR2 receptorstimulation on forskolin-stimulated cAMP accumulation in LM(tk−) cellscould not be reproduced. However, the same LM(tk−) cells yielded areproducible PI hydrolysis response (Table 4), and in independentbinding assays a B_(max) of 4000 fmol/mg protein and a K_(d) of 1.1 nMwhen incubated with porcine ¹²⁵I-galanin. It is concluded that in thecell lines studied thus far, the rat GalR2 is coupled primarily to theactivation of phospholipase C and subsequent inositol phosphatemetabolism, presumably through Gq or a related G protein. The PIresponse was evident as well in LM(tk−) cells stably transfected withthe rat GALR2 receptor cDNA lacking an intron in the coding region(L-rGALR2I-4, see Table 4); membranes from these cells were shown in anindependent experiment to bind porcine ¹²⁵I-galanin with a B_(max) of4800 fmol/mg membrane protein and a K_(d) of 0.2 nM.

The CHO cell line stably transfected with the rat GALR2 receptor(C-rGALR2-79) provided additional detail about the binding and signalingproperties of the receptor. Membranes from stably transfected CHO cellswere bound saturably by porcine ¹²⁵I-galanin with a B_(max) of 520fmol/mg membrane protein and a K_(d) of 0.53 nM. Peptides displaced theporcine ¹²⁵I-galanin (Table 5) with binding affinities similar to thosegenerated from transiently transfected COS-7 cells (Table 1). Receptorstimulation resulted in phosphatidyl inositol hydrolysis (which wasresistant to pre-treatment with pertussis toxin, see FIG. 16D) but hadno effect on cAMP accumulation, again supporting the proposal that therat GALR2 receptor is coupled primarily to phospholipase C activationthrough Gq or a related G protein. Further support for the coupling ofrat GALR2 to Gq is the observation that intracellular free [Ca⁺²] wassubstantially increased by 1 μM rat galanin in C-rGALR2-79(Δ[Ca⁺²]=310±70 nM, n=9) but not in untransfected cells.

It was further demonstrated that rat GALR2 receptor activation could bemonitored by arachidonic acid release (Table 5). Of interest, it wasobserved that the EC₅₀ values from the PI hydrolysis assays were largerthan the K_(i) values from binding assays whereas the EC₅₀ values fromthe arachidonic acid assays were comparable to the binding data. Onepossibility suggested by these data is that the diacylglycerol andcalcium release associated with or induced by inositol phosphatemetabolism leads to activation of protein kinase C, MAP kinase,phospholipase A2 and subsequently to the hydrolysis of arachidonic acidfrom membrane phospholipids. The lower EC₅₀ values in the arachidonicacid assays may reflect an amplification process in the second messengerpathway, such that a maximal arachidonic acid response occurs atsubmaximal inositol phosphate and diacylglycerol or calciumconcentrations.

The stably transfected CHO cells were used to further explore thebinding and signaling properties of the rat GalR2 receptors (Table 6).The peptide binding profile was similar to that generated previouslywith transiently transfected COS-7 cells. Porcine, rat and human galaninbound with high affinity as did C-terminally truncated peptides as shortas galanin 1-12. Chimeric or putative “antagonist”peptides including C7,galantide, M32, M35 and M40 displayed relatively high binding affinityexcept for C7 (Ki=47 nM). Galanin analogs containing D-Trp²(D-Trp²-galanin 1-29 and D-Trp²-galanin 1-16) retained measurablebinding affinity (K_(i)=41 and 110 nM, respectively). The N-terminallytruncated peptide galanin 3-29 was inactive.

Selected peptides were subsequently tested in the arachidonic acidrelease assays. Peptides with measurable EC₅₀ values mimicked themaximal effect of rat galanin (1 μM) on arachidonic acid release andwere classified as full agonists, including C7, galantide, M32, M35 andM40. Relative to rat galanin (EC₅₀=0.67 nM, the endogenous peptidesgalanin (−7) to (+29) and galanin (−9) to (+29), as well as the modifieddi-iodo analog D-Trp2-(3-iodo-L-Tyr9)-(-iodo-L-Tyr26)-galanin, werepotent agonists in the GalR2 functional assay (EC₅₀ values between 2 and4 nM). The binding and functional profiles were in general agreement.Notable exceptions include D-Trp²-galanin 1-29, D-Trp²-galanin 1-16, andC7, all of which generated larger K_(i) values vs. EC₅₀ values; onepossibility is that these peptides were less stable in the binding assayvs. the functional assay. It is, therefore, concluded that thearachidonic acid release assay is useful for assessing peptide potencyand intrinsic activity for the rat GalR2 receptor when stably expressedin CHO cells.

Peptides were further evaluated for their ability to selectivelyactivate the rat GALR1 receptor (monitored in stably transfected LM(tk−)cells using the forskolin-stimulated cAMP accumulation assay) vs. therat GALR2 (monitored in stably transfected CHO cells using thearachidonic acid release assay). Data are reported in Table 7. Relativeto galanin itself, D-Trp2-galanin analogs were selective for GALR2 vs.GALR1. For example, D-Trp2-(3-iodo-L-Tyr9)-(-iodo-L-Tyr26)-galanin was1.5-fold less potent than porcine galanin in the rat GalR2 arachidonicacid assay but 15,000-fold less potent than porcine galanin in the ratGalR1 cAMP assay. D-Trp²-galanin was 8.5-fold less potent than galaninin the rat GALR2 functional assay but >15000-fold less potent thangalanin in the rat GALR1 functional assay. Similarly, D-Trp²-galanin1-16 was 38-fold less potent than galanin in the rat GALR2 functionalassay but >170,000-fold less potent than galanin in the rat GALR1functional assay. It is concluded that D-Trp²-galanin and analogouspeptides may serve as useful tools with which to explore the function ofGALR2 vs. GALR1 receptors in native tissues and physiological systems. Asimilar proposal is made for the N-terminally extended peptides. In thiscase porcine galanin (−7) to (+29) and porcine galanin (−9) to (+29)were 3-fold and 2-fold less potent than porcine galanin in the rat GalR2functional assay respectively, but 350-fold and 400-fold less potentthan porcine galanin in the rat GalR1 functional assay. The N-terminalpeptides can be purified from native tissues and may mediateGALR2-dependent processes in vivo.

The signal transduction pathway outlined for rat GalR2 allows for thedevelopment of functional assays other than inositol phosphate, calcium,and arachidonic acid determination. One such assay relies on the factthat binding of GTP to a G-protein precedes agonist-dependent activationof phospholipase C. we examined whether galanin-dependent activation ofGALR2 could be monitored by the binding of GTPγ³⁵S to membranes fromstably transfected cells. We observed that porcine galanin (1 μM)increased the binding of GTPγ³⁵S from 8,014±113 cpm (n=3) to 10,292±104cpm (n=3) in membranes from C-rGALR2-79 cells. Similarly, porcinegalanin (1 μM) increased the binding of GTPγ³⁵S from 27,545±504 cpm(n=3) to 29,011±526 cpm (n=3) in membranes from L-rGalR2I-4 cells(comprising the intronless plasmid).

Another functional assay relies on the fact that MAP kinasephosphorylation precedes the activation of phospholipase A2. We testedwhether galanin-dependent activation of GALR2 could be monitored bychanges in MAP kinase phosphorylation. Using an antibody specific forphosphorylated MAP kinase in a western blot protocol with achemiluminescent endpoint, we observed an increase in phosphorylated MAPkinase levels when CHO cells expressing either the human or the ratGALR2 receptor were incubated for 10 minutes with human galanin (10 μM).These two assays (GTPγ³⁵S binding and MAP kinase activation) are usefulalternatives to assays described previously and may in some cases haveparticular advantages regarding, for example, speed, throughput,reliability, sensitivity or reagent handling.

Feeding Assays

Rats were injected icv with either galanin, galanin derivatives, orsaline. Cumulative food intake was measured over a period of 3 hours.Baseline food intake associated with the saline control was 1.5 gram. Amaximal food intake of 6.81 grams was observed after a 10 nmoleinjection of galanin. The ED₅₀ for galanin is estimated to be 1 nmole.M40 was also tested in this paradigm. M40 was able to mimic the effectsof galanin, with a maximal food intake of 6.3 grams observed after a 50nmol injection. The ED₅₀ for M40 is estimated to be 20 nmoles. Anotherpeptide, which was found to bind selectively to GALR2 over both GALR3and GALR1, stimulated feeding in rats when administered by i.c.v.injection. This result suggests that GALR2 may mediate galanin-inducedfeeding behavior.

Heterologous Expression of GPCRs in Xenopus Oocytes

Heterologous expression of GPCRs in Xenopus oocytes has been widely usedto determine the identity of signaling pathways activated by agoniststimulation (Gundersen et al., 1983; Takahashi et al., 1987).Application of porcine galanin (100-1000 nM) activates rapid inwardcurrents in 36 of 46 oocytes injected with 5-50 pg rGalR2 mRNA (FIG.13). Equimolar concentrations of C7 induces similar currents whereasgalanin 3-29 is inactive (0/11 oocytes). Oocytes injected with buffer(ND96) alone or 5-HT1a receptor mRNA do not exhibit detectable (<5 nA)responses to galanin (0/19). Current magnitudes in rGalR2 mRNA-injectedoocytes range from small fluctuations of less than 50 nA (excluded fromanalysis) to large rapid currents (up to 3 μA) resembling thoseactivated by stimulation of other receptors (alpha1a receptors—data notshown) that are known to couple to IP3 release and stimulation of Cl-current from the resulting increase in intracellular free Ca⁺⁺(Takahashi et al., 1987). The currents stimulated by galanin in oocytesexpressing rGalR2 are most likely mediated by the endogenouscalcium-activated Cl⁻ channel (Gunderson et al., 1983) because they areblocked in oocytes injected with 50 nl of 10 mM EGTA (5/5) and theydisplay a current-voltage relation that exhibits outward rectificationand a reversal potential of approximately −15 mV (data not shown).

Another functional assay which may be performed using the oocyteexpression system utilizes aequorin. Aequorin, a luminescentphotoprotein isolated from jellyfish, emits blue light upon bindingcalcium. Thus, aequorin is a useful tool to detect expression ofreceptors that trigger the release of intracellular calcium upon agoniststimulation. Application of galanin to oocytes that are injected withaequorin protein alone produces an average response of 1,140 relativelight units (N=4). In contrast, oocytes injected with both rGalR2 mRNAand aequorin protein yield a response of 16,700 relative light units(N=5). Analysis of the time course of luminescence indicates that theoocytes which do not contain rGalR2 mRNA give off a constant, low levelluminescence during the two minute recording interval. However, oocytesexpressing the rGalR2 receptor emit light in a time dependent fashionwith a peak luminescence at about 40 seconds after the addition ofgalanin. This time course is consistent with that seen for other Gprotein-coupled receptors whose expression has been monitored byaequorin.

Receptor Autoradiography

The relative proportion of the total [¹²⁵I]galanin binding attributableto the GALR2 receptor was determined as the binding which was removed by60 nM [D-Trp²]galanin₍₁₋₂₉₎. The numerical representations in Table 2indicate: 1) the relative intensity of the total binding obtained with[¹²⁵I]galanin, with +3 being the maximum; and 2) the relative amount ofthis binding attributable to GALR2, with +3 again being the maximum.

Total [¹²⁵I]galanin binding was observed in many regions of the ratbrain, and was especially intense in the forebrain, including theamygdala, parts of the hypothalamus and thalamus, the septum, and theventral hippocampus. Other regions with intense binding signals includedthe superior colliculus, the central gray, and the dorsal horn of thespinal cord. The inclusion of 5 μM porcine galanin in the incubationresulted in a complete displacement of [¹²⁵I]galanin binding from therat brain tissue sections. The use of 60 nM [D-Trp²]galanin₍₁₋₂₉₎partially displaced [¹²⁵I]galanin binding from many regions of the ratbrain.

The areas most affected by the GALR2 selective ligand were the lateralseptum, the paraventricular hypothalamic nucleus, the centromedial andcentrolateral thalamic nuclei, the amygdalopiriform area of theamygdala, and the superior colliculus. Other forebrain regions withlesser but still significant reductions in [¹²⁵I]galanin bindingincluded the piriform and entorhinal cortices, the globus pallidus, thesupraoptic, lateral, and ventromedial hypothalamic nuclei, and theanterior, cortical, medial, and central amygdaloid nuclei. In themidbrain, pons and medulla, [D-Trp²]galanin₍₁₋₂₉₎ partially reduced thetotal binding in the central gray, the raphe obscurus and raphe magnus,the parabrachial nucleus, the pontine reticular formation, thehypoglossal nucleus, and the gigantocellular reticular nucleus.

In contrast, there were a number of areas in which [D-Trp²]galanin₍₁₋₂₉₎had little or no effect on the total [¹²⁵I]galanin binding. Of these,the most striking were the nucleus of the lateral olfactory tract, theventral hippocampus, and the dorsal horn of the spinal cord. Other areasin which significant binding remained included the olfactory bulb, theinsular cortex, the islands of Calleja, the nucleus accumbens, thelateral habenula, the arcuate nucleus, and the spinal trigeminalnucleus.

Developmental in situ Hybridization

Using oligonucleotide probes, GalR2 mRNA appeared to be developmentallyregulated. At P1 and P5, film autoradiography of the hybridized brainsections revealed clear signals over many thalamic nuclei. In thehypothalamus, both the paraventricular and ventromedial nuclei werelabeled. In addition, the superficial layers of neocortex containedvisible hybridization signal, as did the dorsal hippocampus. In themesencephalon, a low level of hybridization signal was observed in thepretectal region.

Ribonuclease Protection Assay

RNA was isolated and assayed as described from: heart, striated muscle,liver, kidney, and CNS regions. CNS regions included: spinal cord,amygdala, hypothalamus, cerebral cortex, cerebellum, and hippocampus.The highest levels of rGalR2 were detected in the hypothalamus (FIG.14). Lower amounts were found in heart, kidney, hippocampus amygdala,spinal cord, and cerebellum (FIG. 14). mRNA coding for the rGalR2 wasnot detected in RNA extracted from striated muscle or liver.

Generation of Human GALR2 PCR Product

Using PCR primers designed against the fourth and sixth transmembranedomains of the rat GALR2 sequence, NS 525 and NS526, a 300 base pairfragment was amplified from 3 different lots of human genomic DNA.Sequence from all three human genomic DNAs were >98% identical anddisplayed 84% nucleotide identity to the rat GALR2 gene, between thesecond extracellular domain and the 5′ end of the sixth transmembrane.This level of homology is typical of a species homologue relationship inthe GPCR superfamily.

5′ and 3′ RACE Analysis of Human GALR2

5′ RACE was performed on human brain RNA to isolate hGALR2 sequenceupstream of the genomic PCR product above. Using nested reverse primersfrom the fifth transmembrane domain of hGALR2, a 600 base pair fragmentwas amplified. The sequence of this RACE product displayed 91%nucleotide identity to rGALR2 from the 3′ end of the secondtransmembrane domain to the 5′ end of the fifth transmembrane domain.

3′ RACE was performed on human lung RNA to determine the sequence of theCOOH terminus of hGALR2. Using nested forward primers from the fifthtransmembrane domain of hGlR2, a 500 bp RACE product was generated thatshowed a 77% identity to nucleotides 1080-1139 of rGALR2. The sequenceof this RACE product downstream from this region showed less homology torGALR2, and was presumed to represent the COOH terminus and 3′ UT of thehGALR2 gene.

Construction and Screening of a Human Heart cDNA Library

To obtain a full-length hGALR2 clone, superpools of a human heart cDNAlibrary were screened by PCR using primers BB153 and BB169. A 325 basepair fragment was amplified from superpools 6, 9 and 16. Two positiveprimary pools, 69 and 72, were identified from superpool 9, and 1positive primary pool, 121, was identified from superpool 16. Onepositive primary pool, 69, was subdivided into 48 pools of 3333individual clones and screened by PCR. Twelve positive subpools wereidentified and one, 69-11, was subdivided into 20 pools of 1200 clones,plated onto agar plates, and screened by southern analysis. Thirtycolonies that appeared positive were rescreened by PCR using primersBB167 and BB170, revealing 4 positive colonies. One of these, 69-11-5was chosen for further analysis. To evaluate whether this colonyrepresented a single clone, a dilution of the colony was amplified onagar plates and colonies were screened by PCR using primers BB167 andBB170. Five of 20 colonies were positive for hGALR2, indicating that69-11-5 was a mixture of 2 or more clones. One positive colony,69-11-5-3, designated BO29, was amplified as a single hGlR2 clone.Vector-anchored PCR revealed that BO29 is in the correct orientation forexpression, and encodes approximately 200 base pairs of 5′ UT and 5000base pairs 3′ UT. Preliminary single-stranded sequence analysisindicates that BO29 encodes an initiating methionine and a terminationcodon, and contains an intron between the third and fourth transmembranedomains which is approximately 1.2 kb in length. 69-11-5 has beendemonstrated to confer ¹²⁵I galanin binding in transfected COS-7 cells,as assessed by microscopic analysis of photoemulsion-dipped slides. Inaddition, COS-7 cells transfected with the single clone BO29 exhibitsignificant binding of 125I galanin in comparison with COS-7 cellstransfected with control vector. In preliminary radioligand bindingexperiments, ¹²⁵I porcine galanin bound to membranes from COS-7 cellstransfected with BO29, with a specific binding of 4900 fmol/mg, when themembranes (0.005 mg/ml) were incubated with 0.4 nM porcine galanin for30 min. at 30° C. No specific binding was detected to membranes frommock-transfected COS-7 cells when tested under the same conditions.

Human GALR2 Receptor Pharmacology

A human GALR2 receptor construct containing an intron in the codingregion of the cDNA (BO29) was prepared and transiently transfected intoCOS-7 cells. Human GALR2 receptors expressed in the COS-7 cell membraneswere labeled by porcine ¹²⁵I-galanin with an apparent B_(max) of 4200fmol/mg membrane protein and a K_(d) of 0.97 nM. The peptide bindingprofile for the human GALR2 receptor (Table 8) resembled that reportedpreviously for the rat GALR2 in COS-7 cell membranes (Table 1).

A human GALR2 receptor cDNA construct lacking the intron in the codingregion was also prepared (BO39) and transiently transfected into COS-7cells. In a preliminary experiment, membranes from transientlytransfected cells (membrane protein concentration=0.045 mg/ml) wereincubated with porcine ¹²⁵I-galanin (0.17 nM), and specific binding wasmeasured as 480 fmol/mg membrane protein. Assuming an estimated K_(d) of1 nM, the estimated B_(max) for this construct would be ˜3400 fmol/mgmembrane protein. Therefore, it is concluded that the absence of theintron in the coding region of the human GALR2 cDNA has no significanteffect on receptor expression or porcine ¹²⁵I-galanin binding.

Human GALR2 Localization by RT-PCR

RT-PCR was performed on human cDNAs from various brain regions andperipheral tissues. The primers used were BB182 (reverse primer fromhGALR2 stop codon) and BB183 (forward primer from hGalR2 start codon);see methods. The PCR products were Southern blotted and hybridized witha ³²P-labeled oligonucleotide probe (BB247). Of the tissues examined,the highest levels were found in the heart and kidney, with high levelsalso detected in hypothalamus, liver and small intestine. Moderatelevels were detected in hippocampus, and somewhat lower levels werefound in the retina. No signal was detected in cerebral cortex, adrenalgland, lung, lymph node, spleen or stomach.

Experimental Discussion

In order to clone additional members of the galanin receptor family, anexpression cloning strategy based on the potential presence of multiplegalanin receptors in the hypothalamus was designed. Using this strategya cDNA clone encoding a galanin receptor from rat hypothalamus, termedGALR2, was isolated that is distinct from the previously cloned GALR1receptors.

Transient transfection of the isolated cDNA (K985) encoding GALR2resulted in high affinity binding of [¹²⁵I]-porcine galanin. The highbinding affinity of the GALR2 receptor for galanin₍₁₋₂₉₎ and itstruncated analogs galanin₍₁₋₁₆₎ and galanin₍₁₋₁₅₎ strongly supports thenotion that the GALR2 receptor is a novel galanin receptor subtype. Boththe rat GALR1 and GALR2 receptors seem to bind preferentially to theamino terminus of galanin. Deletion of 13 or 14 amino acids from thecarboxyl terminus of galanin still yields peptides with high bindingaffinity at both the GALR1 and GALR2 receptors. Furthermore, thetruncation of the first two amino acids of the amino terminus led to acomplete loss of affinity at both GALR1 and GALR2. Consistent with thisnotion are the findings that the chimeric peptides, which shareidentical amino acid sequences in the first 12 amino acids with galaninhad very similar binding affinities for either GALR1 or GALR2 receptors.In spite of these similarities, the substitution of L-tryptophan withD-tryptophan in position 2 of porcine galanin₍₁₋₂₉₎([D-Trp²]galanin₍₁₋₂₉₎ led to a 7,000-fold loss in affinity at the GALR1receptor compared to only a 14-fold reduction at the GALR2 receptor. Thesame substitution in the truncated analog galanin₍₁₋₆₎ led to a4,200-fold reduction in affinity at the GALR1 receptor, and only a6-fold reduction in affinity at the GALR2 receptor. Relative to porcinegalanin, D-Trp2-(3-iodo-L-Tyr9)-(-iodo-L-Tyr26)-galanin was much morepotent as a GalR2 agonist (only 1.5-fold less potent in arachidonic acidassays) than as a GalR1 agonist (15,000-fold less potent in cAMP assays;see Table 7). These data suggest that galanin analogs, withmodifications at the 2-position, are better tolerated at the GALR2receptor than at the GALR1 receptor as long as the side chain is anaromatic moiety. The data further suggest that the D-Trp2-galaninanalogs may be used as tools to differentiate GalR2 from GalR1-dependentprocesses.

Conversely, the substitution of tyrosine with alanine in position 9 ofgalanin₍₁₋₁₆₎, (i.e., to make [Ala]⁹ galanin) leads to a 680-foldreduction in affinity at the GALR1 receptor and to a 60,000-foldreduction in affinity at the GALR2 receptor. Altogether, the majordifferences in binding selectivity of the substituted analogs of galaninsuggest the existence of substantial differences in the binding domainsof these two receptor subtypes.

The existence of such structural differences between the GALR1 and GALR2receptors are indicative of the potential for the design and discoveryof novel subtype selective compounds. In this regard, the expression ofthe cDNA encoding the rat GALR2 receptors in cultured cell linesprovides a unique tool for the discovery of therapeutic agents targetedat galanin receptors.

Localization of Galanin Receptors

The high affinity of [D-Trp²]galanin₍₁₋₂₉₎ for the cloned GALR2 receptor(6 nM), and its low affinity for the GALR1 receptor (3 μM), makes it auseful tool for receptor autoradiographic studies. Thus, brain areas inwhich the total [¹²⁵I]galanin binding is significantly reduced by[D-Trp²]galanin₍₁₋₂₉₎ are interpreted as areas containing a highproportion of GALR2 receptors, or other galanin receptors with similarhigh affinity for [D-Trp²]galanin₍₁₋₂₉₎. Those with lesser reductionsare seen as regions containing a higher concentration of GALR1receptors. The lateral septum, the paraventricular hypothalamic nucleus,the centromedial and centrolateral thalamic nuclei, the amygdalopiriformarea of the amygdala, and the superior colliculus all appear to containprimarily GALR2 receptors. In contrast, the nucleus of the lateralolfactory tract, the ventral hippocampus, and the dorsal horn of thespinal cord appear to contain primarily GALR1 receptors. Thepredominance of the GALR1 receptor in these regions is consistent withpublished reports of the GALR1 messenger RNA localization (Parker etal., 1995; Gustafson et al., 1996). In most other regions, there appearsto be a significant overlap between the two subtypes.

While the functional implications of the GALR2 receptor localization arenot well understood at present, there are a number of physiologicalprocesses attributable to galanin that could be mediated by thisreceptor. These include feeding (paraventricular hypothalamic nucleus),cognition (septum and hippocampus) , analgesia and/or sensory processing(midline thalamic nuclei), and anxiety and depression (amygdala andhypothalamus).

The observation that galanin is co-released with norepinephrine fromsympathetic nerve terminals suggests that galanin could act via galaninreceptors in the periphery to modulate nearly every physiologicalprocess controlled by sympathetic innervation. Additional therapeuticindications not directly related to localization (supra) includediabetes, hypertension, cardiovascular disorders, regulation of growthhormone release, regulation of fertility, gastric ulcers,gastrointestinal motility/transit/absorption/secretion, glaucoma,inflammation, immune disorders, respiratory disorders (eg. asthma,emphysema).

The physiological and anatomical distribution of galanin-containingneurons suggests potential roles of galanin receptors mediating effectson cognition, analgesia, neuroendocrine regulation, control of insulinrelease and control of feeding behavior. Of particular relevance to therole of the novel GALR2 receptor, are those functions mediated bygalanin receptors in the rat hypothalamus.

Studies in rats indicate that the injection of galanin in thehypothalamus increases food intake (Kyrouli et al, 1990, and Schick etal, 1993) and that this stimulatory effect of galanin is blocked byprior administration of M40 and C7 (Liebowitz and Kim, 1992; and Corwin,1993). The expression of the mRNA encoding the GALR1 receptor in the rathypothalamus, (Parker et al., 1995, Gustafson et al., 1996) and the factthat the novel GALR2 receptor was cloned from a cDNA library preparedfrom rat hypothalamus argues in favor of either receptor subtype to beinvolved in the regulation of feeding behavior (Parker et al., 1996).However, the evidence against the involvement of GALR1 in thestimulation of feeding behavior stems from the fact that M40 and C7 areknown to be agonists, and not antagonists, in cell lines expressing thecloned human and rat GALR1 receptors (Heuillet et al. 1994; Hale et al.1993; and Bartfai et al. 1993).

The distribution of GALR2 mRNA in the rat brain and periphery has beendetermined by ribonuclease protection assay, in situ hybridization,Northern blot analysis and RT-PCR. The results of these studies suggestthat this receptor is potentially involved in mediating many of thephysiological roles ascribed to the peptide galanin. In the adult rat,localization of the GalR2 mRNA in the hypothalamus indicates a role forthis receptor in homeostatic mechanisms, including food intake andneuroendocrine regulation. The presence of GALR2 mRNA in the neocortexand dorsal hippocampus suggest an involvement in cognition, which isconsistent with documented changes in galanin and galanin receptorexpression during aging and in the brains of Alzheimer's patients(Chan-Palay, 1988; Leverenz et al., 1996) Galanin also hasantinociceptive effects, and the localization of GALR2 mRNA in thespinal cord (present investigation) and dorsal root ganglia (O'Donnellet al., 1996) implicate this receptor in pain neurotransmission. Thelocalization of GALR2 mRNA in the cerebellum is intriguing, as itsuggests a role for galanin and the GalR2 receptor in planned movementsand potentially in movement disorders.

In addition to the localization observed in adult animals, it alsoappears that the GALR2 mRNA is developmentally regulated, with thehighest levels observed early in postnatal development. Thus, it ispossible that this galanin receptor plays a role in developmentalprocesses which occur during the first postnatal week, such as axonalguidance and synapse formation.

A unique pharmacological profile for the GALR2 receptor has beengenerated through binding and functional assays. This profile can beused to deduce the physiological function of the GalR2 receptor in vivo.Consider the agonist activity of galanin 1-16, for example. Galanin 1-16is reported to function as an agonist in various models of hypothalamic,pituitary and pancreatic function (Kask et al.) Galanin 1-16 is alsoreported to mimic the effects of galanin on the flexor reflex in therat. The N-terminally extended peptides galanin (−7) to (+) 29 andgalanin (−9) to (+) 29, also characterized as rat GALR2 agonists, canmimic the effects of galanin in a rat flexor reflex assay (Weisenfeld).Taken together, these data suggest a potential role for the rat GalR2receptor in a range of physiology or pathophysiology including diabetes,pain, reproduction, obesity and eating disorders.

The agonist activity of M40 in GALR2 in vitro assays is particularlyintriguing when viewed in the context of behavioral feeding models. Fromthe literature, one might conclude that the agonist activity of M40 invitro is in apparent conflict with the antagonist activity reported forM40 in behavioral models of food intake, and that the GALR2 receptor istherefore unlikely to mediate the feeding response. The data generatedand reported in the subject application do not support this conclusion.Rather, the data from behavioral feeding models indicate that M40 is anorexigenic peptide whose maximal effect is comparable to that forgalanin itself. The agonist activity reported herein for M40 both invitro and in vivo is consistent with the proposal that the rat GALR2receptor mediates the stimulatory effect of galanin on food intake inthe central nervous system. These data further suggest that the ratGALR2 receptor represents a target for the design of therapeuticcompounds for the treatment of obesity and related disorders.

TABLE 1 Binding of galanin peptide analogs to the recombinant rat GALR1and GALR2 receptors transiently expressed in COS 7 cells. GALR1 (pKi)GALR2 (pKi) Analog Mean SEM* Mean SEM porcine galanin ₍₁₋₂₉₎ 9.34 0.159.35 0.14 [D-Trp²] porcine 5.46 0.04 8.19 0.26 galanin ₍₁₋₂₉₎ [Phe²]porcine 5.99 0.13 5.64 0.11 galanin ₍₁₋₂₉₎ [D-Ala⁷] porcine 8.66 0.048.76 0.09 galanin ₍₁₋₂₉₎ galanin ₍₁₋₁₆₎ 8.66 0.01 8.76 0.13 [D-Trp²]galanin ₍₁₋₁₆₎ 4.40 0.09 8.02 0.10 [Ala⁵] galanin ₍₁₋₁₆₎ 6.27 0.05 7.460.13 [Ala⁹] galanin ₍₁₋₁₆₎ 5.83 0.02 3.98 0.10 galanin ₍₁₋₁₅₎ 8.47 0.049.19 0.06 [Phe²] galanin ₍₁₋₁₅₎ 4.63 0.03 5.85 0.49 porcine galanin₍₃₋₂₉₎ <4.0 <4.0 galantide 8.02 0.08 8.70 0.07 C-7 7.79 0.01 7.72 0.09M32 9.21 0.10 9.23 0.05 M35 9.48 0.07 9.24 0.10 M40 8.44 0.09 9.14 0.21*SEM= standard error of the mean, from 3 independent experiments.

TABLE 2 Distribution of [¹²⁵I] galanin binding in rat brain. Totalbinding is compared to the amount attributable to GALR2 (as indicated bydisplacement of [¹²⁵I] galanin by 60 nM [D-Trp²] porcine galanin₍₁₋₂₉₎).Total Putative [¹²⁵I] Gal GALR2 Potential Region binding sitesApplications Olfactory bulb +3 +1 Modulation of olfactory sensationAnterior olfactory n. +3 +1 Modulation of olfactory sensation Cortexdorsal neocortex, +1 +1 Sensory layer 4 integration piriform +2 +1Modulation of olfactory sensation agranular insular +3 +1 Processing ofvisceral information entorhinal +2 +1 dorsal endopiriform +2 +1Claustrum +2 +1 Visual processing Basal ganglia n. accumbens +2 0Modulation of olfactory tubercle +2 +1 dopaminergic globus pallidus +1+1 function islands of Calleja Septal area lateral septum +3 +2Cognitive diagonal band n. +2 0 enhancement via cholinergic systemHypothalamus anterior +1 0 Neuroendocrine regulation supraoptic n. +2paraventricular +2 +2 Appetite/obesity ventromedial +2 +1 arcuate +1 0lateral +2 +1 medial mammillary +2 +1 Thalamus paraventricular n. +1 0Analgesia/sensory centromedial +3 +2 modulation paracentral +3 +1rhomboid +1 0 reuniens +2 +1 mediodorsal +2 0 reticular n. +1 +½centrolateral n. +3 +2 zona incerta +2 +1 lateral dorsal +1 +½ habenula+3 +1 Anxiety/sleep disorders Hippocampus Cal, ventral +3 0 Cognitionsubiculum +2 +1 enhancement/ ischaemia Amygdala bed n. stria +3 +1Anxiolytic, terminalis appetite, n. lateral olfactory +3 0 depressiontract Amygdala anterior +2 +1 Anxiolytic, medial +3 +1 appetite,cortical +2 +1 depression central +3 +1 amygdalohippocampal +2 0amygdalopiriform +3 +2 Midbrain superior colliculus +3 +2 Visualfunction raphe obscurus +2 +1 Analgesia central gray +2 +1 AnalgesiaPons/medulla raphe magnus +2 +1 Analgesia parabrachial n. +2 +1 pontineret. n. +2 +1 reticulotegmental +2 +1 gigantocellular +2 motortrigeminal +1 0 spinal trigeminal +3 Migraine hypoglossal n. +2 +1 Motorcoordination area postrema +1 0 Spinal cord dorsal horn +3 +1 Analgesia

TABLE 3 Northern blot hybridization of GALR2 receptor in brain andvarious peripheral rat tissues. Mean Tissue Blot 1 Blot 2 SignalTherapeutic Indications Heart +++ ++ 2.5 Cardiovascular Indications(including hypertension and heart failure) Brain ++++ ++++ 4.0Obesity/feeding, analgesia, cognition enhancement, Alzheimer's disease,depression, anxiety, sleep disorders, parkinson's disease, traumaticbrain injury, convulsion/epilepsy Spleen ++ ++ 2.0 Immune functions,hematopoiesis Lung ++++ ++++ 4.0 Respiratory disorders, asthma,emphysema, lung cancer diagnostics Liver ++ — 1.0 Diabetes Skeletal + ++1.5 Diabetes Muscle Kidney +++ +++ 3.0 Hypertension, electrolytebalance, diuretic, anti-diuretic Testis +++ + 2.0 Reproductive function

TABLE 4 Inositol phosphate hydrolysis in LM(tk-) cells stablytransfected with GALR2. EC₅₀ PI (nM) EC₅₀ PI (nM) rat GALR2 L-rGALR2I-4Peptide (with intron) (intronless) porcine galanin 21 14 M35 29 28D-Trp²-galanin 1-16 1380 660 D-Trp²-galanin 1-29 200 230 galanin 1-16 6518 M40 28 47 M32 13 35

TABLE 5 Rat GALR2 receptors stably transfected in CHO Comparison ofbinding data, phosphatidyl inositol release, and arachidonic acidrelease in C-rGalR2-79. K_(1 from) porcine EC₅₀ from EC₅₀ from¹²⁵I-galanin PI arachi- binding hydrolysis donic acid assays assaysassays Peptide (nM) (nM) (nM) rat galanin 0.52 14 0.67 porcine galanin0.94 15 1.3 porcine galanin 3.5 91 2.6 1-16 D-Trp²-galanin 110 590 501-16 D-Trp²-(3-iodo- — 580 2.0 L-Tyr9)-(3-iodo- L-Tyr26)-galanin porcinegalanin — 220 3.9 (−7) to (+) 29 porcine galanin — — 2.8 (−9) to (+) 29porcine galanin 1.5 200 2.3 (1-15) porcine galanin >1000 >1000 >1000(3-29) galantide 4.9 11 0.93 C7 48 11 2.4 M32 5.6 14 2.5 M35 3.4 44 1.3M40 3.5 58 2.7

TABLE 6 CHO GALR2 pharmacology: binding (K_(i) vs. ¹²⁵I porcine galanin)vs. function (arachidonic acid hydrolysis) Rat GALR2 Rat GALR2 K_(i),EC₅₀ AA, C-rGalR2-79 C-rGalR2-79 Peptide (nM) (nM) Human galanin 1.2Porcine galanin 0.94 1.3 rat galanin 0.52 0.67 porcine gal −7 to +29 3.0porcine galanin −9 to +29 4.0 porcine galanin 3-iodo-L- 0.8 Tyr9-galaninporcine galanin 3-iodo-L- 1.0 Tyr26 galanin porcine Phe2-galanin >1000porcine D-Trp2-galanin 41 11 D-Trp2-3-iodo-L-Tyr9- 3.0 galanin porcineD-Trp2-3-iodo-L- 6.0 Tyr26-galanin D-Trp2-(3-iodo-L-Tyr9)-(3- 2.0iodo-L-Tyr26)-galanin D-Ala7-galanin 6.2 5.4 porcine galanin3-29 >1000 >1000 porcine galanin 9-29 >1000 >1000 porcine galanin17-29 >1000 porcine galanin 1-16 3.5 2.6 porcine Ala2-galanin 1-16 >1000porcine D-Trp2-galanin 1-16 110 50 porcine Ala5-galanin 1-16 >620porcine Ala9-galanin 1-16 >1000 porcine galanin 1-15 1.5 2.3Phe2-galanin 1-16 >1000 >1000 porcine galanin 1-12 2.1 2.3 porcinegalanin 1-9 >1000 >1000 C7 48 2.4 Galantide 4.9 0.93 M32 3.4 2.5 M35 5.81.3 M40 3.5 2.7

TABLE 7 Peptide-dependent activation of rat GALR1 vs. rat GALR2. LM(tk-)C-RGalR2-79 Rat GALR1 arachidonic acid cAMP assay assay Peptide EC₅₀(nM) EC₅₀ (nM) Porcine galanin 0.06 1.3 rat galanin 0.05 0.67 porcineD-Trp²-galanin >850 11 porcine galanin 3-29 >1000 >1000 porcine galanin1-16 0.34 2.6 porcine >1000 50 D-Trp²-galanin 1-16 C7 0.52 2.4 Galantide0.08 0.93 M32 0.34 2.5 M35 0.15 1.3 M40 0.82 2.7 D-Trp2-(3-iodo-L- 9002.0 Tyr9)-(3-iodo-L- Tyr26)-galanin porcine galanin (−7) 21 3.9 to (+29)porcine galanin (−9) 24 2.8 to (+29)

TABLE 8 Peptide binding profile: Human GALR2 vs. rat GALR2 transientlyexpressed in COS-7 Human GALR2 K_(i) Rat GALR2 K_(i) Peptide (nM) (nM)porcine galanin 1-16 15  7.2*  porcine galanin 0.72  0.45  M40 5.3 0.72  porcine D-Trp²- 290 52*   galanin M32 7.9 12*   rat galanin 1.0 0.52* *additional experiments were performed with some of the peptidesshown in Table 1.

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32 24 base pairs nucleic acid single linear DNA 1 TGGGCAACAG CCTAGTGATCACCG 24 24 base pairs nucleic acid single linear DNA 2 CTGCTCCCAGCAGAAGGTCT GGTT 24 24 base pairs nucleic acid single linear DNA 3CCTCAGTGAA GGGAATGGGA GCGA 24 26 base pairs nucleic acid single linearDNA 4 CTCATTGCAA ACACGGCACT TGAACA 26 24 base pairs nucleic acid singlelinear DNA 5 CTTGCTTGTA CGCCTTCCGG AAGT 24 24 base pairs nucleic acidsingle linear DNA 6 GAGAACTTCA TCACGCTGGT GGTG 24 1119 base pairsnucleic acid single linear DNA CDS 1..1119 7 ATG AAT GGC TCC GGC AGC CAGGGC GCG GAG AAC ACG AGC CAG GAA GGC 48 Met Asn Gly Ser Gly Ser Gln GlyAla Glu Asn Thr Ser Gln Glu Gly 1 5 10 15 GGT AGC GGC GGC TGG CAG CCTGAG GCG GTC CTT GTA CCC CTA TTT TTC 96 Gly Ser Gly Gly Trp Gln Pro GluAla Val Leu Val Pro Leu Phe Phe 20 25 30 GCG CTC ATC TTC CTC GTG GGC ACCGTG GGC AAC GCG CTG GTG CTG GCG 144 Ala Leu Ile Phe Leu Val Gly Thr ValGly Asn Ala Leu Val Leu Ala 35 40 45 GTG CTG CTG CGC GGC GGC CAG GCG GTCAGC ACC ACC AAC CTG TTC ATC 192 Val Leu Leu Arg Gly Gly Gln Ala Val SerThr Thr Asn Leu Phe Ile 50 55 60 CTC AAC CTG GGC GTG GCC GAC CTG TGT TTCATC CTG TGC TGC GTG CCT 240 Leu Asn Leu Gly Val Ala Asp Leu Cys Phe IleLeu Cys Cys Val Pro 65 70 75 80 TTC CAG GCC ACC ATC TAC ACC CTG GAC GACTGG GTG TTC GGC TCG CTG 288 Phe Gln Ala Thr Ile Tyr Thr Leu Asp Asp TrpVal Phe Gly Ser Leu 85 90 95 CTC TGC AAG GCT GTT CAT TTC CTC ATC TTT CTCACT ATG CAC GCC AGC 336 Leu Cys Lys Ala Val His Phe Leu Ile Phe Leu ThrMet His Ala Ser 100 105 110 AGC TTC ACG CTG GCC GCC GTC TCC CTG GAC AGGTAT CTG GCC ATC CGC 384 Ser Phe Thr Leu Ala Ala Val Ser Leu Asp Arg TyrLeu Ala Ile Arg 115 120 125 TAC CCG CTG CAC TCC CGA GAG TTG CGC ACA CCTCGA AAC GCG CTG GCC 432 Tyr Pro Leu His Ser Arg Glu Leu Arg Thr Pro ArgAsn Ala Leu Ala 130 135 140 GCC ATC GGG CTC ATC TGG GGG CTA GCA CTG CTCTTC TCC GGG CCC TAC 480 Ala Ile Gly Leu Ile Trp Gly Leu Ala Leu Leu PheSer Gly Pro Tyr 145 150 155 160 CTG AGC TAC TAC CGT CAG TCG CAG CTG GCCAAC CTG ACA GTA TGC CAC 528 Leu Ser Tyr Tyr Arg Gln Ser Gln Leu Ala AsnLeu Thr Val Cys His 165 170 175 CCA GCA TGG AGC GCA CCT CGA CGT CGA GCCATG GAC CTC TGC ACC TTC 576 Pro Ala Trp Ser Ala Pro Arg Arg Arg Ala MetAsp Leu Cys Thr Phe 180 185 190 GTC TTT AGC TAC CTG CTG CCA GTG CTA GTCCTC AGT CTG ACC TAT GCG 624 Val Phe Ser Tyr Leu Leu Pro Val Leu Val LeuSer Leu Thr Tyr Ala 195 200 205 CGT ACC CTG CGC TAC CTC TGG CGC ACA GTCGAC CCG GTG ACT GCA GGC 672 Arg Thr Leu Arg Tyr Leu Trp Arg Thr Val AspPro Val Thr Ala Gly 210 215 220 TCA GGT TCC CAG CGC GCC AAA CGC AAG GTGACA CGG ATG ATC ATC ATC 720 Ser Gly Ser Gln Arg Ala Lys Arg Lys Val ThrArg Met Ile Ile Ile 225 230 235 240 GTG GCG GTG CTT TTC TGC CTC TGT TGGATG CCC CAC CAC GCG CTT ATC 768 Val Ala Val Leu Phe Cys Leu Cys Trp MetPro His His Ala Leu Ile 245 250 255 CTC TGC GTG TGG TTT GGT CGC TTC CCGCTC ACG CGT GCC ACT TAC GCG 816 Leu Cys Val Trp Phe Gly Arg Phe Pro LeuThr Arg Ala Thr Tyr Ala 260 265 270 TTG CGC ATC CTT TCA CAC CTA GTT TCCTAT GCC AAC TCC TGT GTC AAC 864 Leu Arg Ile Leu Ser His Leu Val Ser TyrAla Asn Ser Cys Val Asn 275 280 285 CCC ATC GTT TAC GCT CTG GTC TCC AAGCAT TTC CGT AAA GGT TTC CGC 912 Pro Ile Val Tyr Ala Leu Val Ser Lys HisPhe Arg Lys Gly Phe Arg 290 295 300 AAA ATC TGC GCG GGC CTG CTG CGC CCTGCC CCG AGG CGA GCT TCG GGC 960 Lys Ile Cys Ala Gly Leu Leu Arg Pro AlaPro Arg Arg Ala Ser Gly 305 310 315 320 CGA GTG AGC ATC CTG GCG CCT GGGAAC CAT AGT GGC AGC ATG CTG GAA 1008 Arg Val Ser Ile Leu Ala Pro Gly AsnHis Ser Gly Ser Met Leu Glu 325 330 335 CAG GAA TCC ACA GAC CTG ACA CAGGTG AGC GAG GCA GCC GGG CCC CTT 1056 Gln Glu Ser Thr Asp Leu Thr Gln ValSer Glu Ala Ala Gly Pro Leu 340 345 350 GTC CCA CCA CCC GCA CTT CCC AACTGC ACA GCC TCG AGT AGA ACC CTG 1104 Val Pro Pro Pro Ala Leu Pro Asn CysThr Ala Ser Ser Arg Thr Leu 355 360 365 GAT CCG GCT TGT TAA 1119 Asp ProAla Cys * 370 372 amino acids amino acid linear protein 8 Met Asn GlySer Gly Ser Gln Gly Ala Glu Asn Thr Ser Gln Glu Gly 1 5 10 15 Gly SerGly Gly Trp Gln Pro Glu Ala Val Leu Val Pro Leu Phe Phe 20 25 30 Ala LeuIle Phe Leu Val Gly Thr Val Gly Asn Ala Leu Val Leu Ala 35 40 45 Val LeuLeu Arg Gly Gly Gln Ala Val Ser Thr Thr Asn Leu Phe Ile 50 55 60 Leu AsnLeu Gly Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro 65 70 75 80 PheGln Ala Thr Ile Tyr Thr Leu Asp Asp Trp Val Phe Gly Ser Leu 85 90 95 LeuCys Lys Ala Val His Phe Leu Ile Phe Leu Thr Met His Ala Ser 100 105 110Ser Phe Thr Leu Ala Ala Val Ser Leu Asp Arg Tyr Leu Ala Ile Arg 115 120125 Tyr Pro Leu His Ser Arg Glu Leu Arg Thr Pro Arg Asn Ala Leu Ala 130135 140 Ala Ile Gly Leu Ile Trp Gly Leu Ala Leu Leu Phe Ser Gly Pro Tyr145 150 155 160 Leu Ser Tyr Tyr Arg Gln Ser Gln Leu Ala Asn Leu Thr ValCys His 165 170 175 Pro Ala Trp Ser Ala Pro Arg Arg Arg Ala Met Asp LeuCys Thr Phe 180 185 190 Val Phe Ser Tyr Leu Leu Pro Val Leu Val Leu SerLeu Thr Tyr Ala 195 200 205 Arg Thr Leu Arg Tyr Leu Trp Arg Thr Val AspPro Val Thr Ala Gly 210 215 220 Ser Gly Ser Gln Arg Ala Lys Arg Lys ValThr Arg Met Ile Ile Ile 225 230 235 240 Val Ala Val Leu Phe Cys Leu CysTrp Met Pro His His Ala Leu Ile 245 250 255 Leu Cys Val Trp Phe Gly ArgPhe Pro Leu Thr Arg Ala Thr Tyr Ala 260 265 270 Leu Arg Ile Leu Ser HisLeu Val Ser Tyr Ala Asn Ser Cys Val Asn 275 280 285 Pro Ile Val Tyr AlaLeu Val Ser Lys His Phe Arg Lys Gly Phe Arg 290 295 300 Lys Ile Cys AlaGly Leu Leu Arg Pro Ala Pro Arg Arg Ala Ser Gly 305 310 315 320 Arg ValSer Ile Leu Ala Pro Gly Asn His Ser Gly Ser Met Leu Glu 325 330 335 GlnGlu Ser Thr Asp Leu Thr Gln Val Ser Glu Ala Ala Gly Pro Leu 340 345 350Val Pro Pro Pro Ala Leu Pro Asn Cys Thr Ala Ser Ser Arg Thr Leu 355 360365 Asp Pro Ala Cys 370 2200 base pairs nucleic acid single linear DNACDS 46..414 CDS 1422..2171 9 CAAGACCCGG ACAGCTGCGG GAGCGGCGTC CACTTTGGTGATACC ATG AAT GGC 54 Met Asn Gly 1 TCC GGC AGC CAG GGC GCG GAG AAC ACGAGC CAG GAA GGC GGT AGC GGC 102 Ser Gly Ser Gln Gly Ala Glu Asn Thr SerGln Glu Gly Gly Ser Gly 5 10 15 GGC TGG CAG CCT GAG GCG GTC CTT GTA CCCCTA TTT TTC GCG CTC ATC 150 Gly Trp Gln Pro Glu Ala Val Leu Val Pro LeuPhe Phe Ala Leu Ile 20 25 30 35 TTC CTC GTG GGC ACC GTG GGC AAC GCG CTGGTG CTG GCG GTG CTG CTG 198 Phe Leu Val Gly Thr Val Gly Asn Ala Leu ValLeu Ala Val Leu Leu 40 45 50 CGC GGC GGC CAG GCG GTC AGC ACC ACC AAC CTGTTC ATC CTC AAC CTG 246 Arg Gly Gly Gln Ala Val Ser Thr Thr Asn Leu PheIle Leu Asn Leu 55 60 65 GGC GTG GCC GAC CTG TGT TTC ATC CTG TGC TGC GTGCCT TTC CAG GCC 294 Gly Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val ProPhe Gln Ala 70 75 80 ACC ATC TAC ACC CTG GAC GAC TGG GTG TTC GGC TCG CTGCTC TGC AAG 342 Thr Ile Tyr Thr Leu Asp Asp Trp Val Phe Gly Ser Leu LeuCys Lys 85 90 95 GCT GTT CAT TTC CTC ATC TTT CTC ACT ATG CAC GCC AGC AGCTTC ACG 390 Ala Val His Phe Leu Ile Phe Leu Thr Met His Ala Ser Ser PheThr 100 105 110 115 CTG GCC GCC GTC TCC CTG GAC AGG TGAGTGAACATCGGAGAACT ATTGTATCTG 444 Leu Ala Ala Val Ser Leu Asp Arg 120AGATAGGGGCTTGGGCTGGAGTCACTACACAGGGGATCCAGAAGGCATGAGCAGAATGGG 504CGAGAACACTGAAATTACAAAGTGGCCTGAGGCCGTGAAACGCAAGGGGGAGGGAGATTA 564AGACTCAGTGACTGAGAGTGTCTAAGTCGATGGGAGAAATCGGGTCTCTGGGGTCCTCGC 624ATTATTACTGCTTGAGTTAAATGTCTCTGTGAAACATTGCAGTTCTCAGGCCAGAGTTGG 684CAGGAAAAGTAACTCGCCAGTGTTCAGATGCTGTTTGAGAGCTGCAGAGAAGCATCTGCT 744TCTTAGCACCAAGCTCAGCACCTGGGGCGTTGTCCGGCGCCTTAGGCTTAGGACTGGGCT 804GTGCTGTGTTAAGACCCATGCTCAAGTCCAACGGAGTGTAAGCGAGGGCTCCTAGCTGAC 864ACCCAGAGCCCTCCAGGCCAAGGCTCCCCTCACCGAGATGCCAGCGGTTTTATGCTCCTT 924CCATAGGTAAAGGACCCAGAAAGAAACATCCAGTATGCCCGGAGGGATCTTGACTGGAAA 984AGACTGAATCCTGGTCTGGTGACCTTAGTTCCCTGCCCTTTCACATCACTTGGACATTCC 1044CACAGAAGAGCGGTGAAGAGGCGGTGGTCCTTATTCTCCTCTGGTTTCCACTGAGTGCAA 1104CATGTGCGTCCTGAGTACGCTGGAGGGACTCACAAAATTTCAGCTTTCTT TAGGAGTTTC 1164CTTGCTGTAGTTTGACCCAAGTCTTCTCCAGGTTTCTGTCAGAACCTCAG GCATGAGGGA 1224TCTGCCTCCCCTGGTTGTCACCAGAGGATAACAATCACTGCCCCCAGAAATCCAGACAGA 1284TTCTACAACTTTTAGTCTTCGGTGTTTTGGGGGTGCCCCTTCACGTGGAG TAGGTCGGTG 1344GCCACATTCCCAGGAGTGACAATAGCCTAGCAGTGAATCCTCTCGCTTAG CTGATGCCCC 1404CCCACTGTCCCCACAGG TAT CTG GCC ATC CGC TAC CCG CTG CAC TCC CGA 1454 TyrLeu Ala Ile Arg Tyr Pro Leu His Ser Arg 1 5 10 GAG TTG CGC ACA CCT CGAAAC GCG CTG GCC GCC ATC GGG CTC ATC TGG 1502 Glu Leu Arg Thr Pro Arg AsnAla Leu Ala Ala Ile Gly Leu Ile Trp 15 20 25 GGG CTA GCA CTG CTC TTC TCCGGG CCC TAC CTG AGC TAC TAC CGT CAG 1550 Gly Leu Ala Leu Leu Phe Ser GlyPro Tyr Leu Ser Tyr Tyr Arg Gln 30 35 40 TCG CAG CTG GCC AAC CTG ACA GTATGC CAC CCA GCA TGG AGC GCA CCT 1598 Ser Gln Leu Ala Asn Leu Thr Val CysHis Pro Ala Trp Ser Ala Pro 45 50 55 CGA CGT CGA GCC ATG GAC CTC TGC ACCTTC GTC TTT AGC TAC CTG CTG 1646 Arg Arg Arg Ala Met Asp Leu Cys Thr PheVal Phe Ser Tyr Leu Leu 60 65 70 75 CCA GTG CTA GTC CTC AGT CTG ACC TATGCG CGT ACC CTG CGC TAC CTC 1694 Pro Val Leu Val Leu Ser Leu Thr Tyr AlaArg Thr Leu Arg Tyr Leu 80 85 90 TGG CGC ACA GTC GAC CCG GTG ACT GCA GGCTCA GGT TCC CAG CGC GCC 1742 Trp Arg Thr Val Asp Pro Val Thr Ala Gly SerGly Ser Gln Arg Ala 95 100 105 AAA CGC AAG GTG ACA CGG ATG ATC ATC ATCGTG GCG GTG CTT TTC TGC 1790 Lys Arg Lys Val Thr Arg Met Ile Ile Ile ValAla Val Leu Phe Cys 110 115 120 CTC TGT TGG ATG CCC CAC CAC GCG CTT ATCCTC TGC GTG TGG TTT GGT 1838 Leu Cys Trp Met Pro His His Ala Leu Ile LeuCys Val Trp Phe Gly 125 130 135 CGC TTC CCG CTC ACG CGT GCC ACT TAC GCGTTG CGC ATC CTT TCA CAC 1886 Arg Phe Pro Leu Thr Arg Ala Thr Tyr Ala LeuArg Ile Leu Ser His 140 145 150 155 CTA GTT TCC TAT GCC AAC TCC TGT GTCAAC CCC ATC GTT TAC GCT CTG 1934 Leu Val Ser Tyr Ala Asn Ser Cys Val AsnPro Ile Val Tyr Ala Leu 160 165 170 GTC TCC AAG CAT TTC CGT AAA GGT TTCCGC AAA ATC TGC GCG GGC CTG 1982 Val Ser Lys His Phe Arg Lys Gly Phe ArgLys Ile Cys Ala Gly Leu 175 180 185 CTG CGC CCT GCC CCG AGG CGA GCT TCGGGC CGA GTG AGC ATC CTG GCG 2030 Leu Arg Pro Ala Pro Arg Arg Ala Ser GlyArg Val Ser Ile Leu Ala 190 195 200 CCT GGG AAC CAT AGT GGC AGC ATG CTGGAA CAG GAA TCC ACA GAC CTG 2078 Pro Gly Asn His Ser Gly Ser Met Leu GluGln Glu Ser Thr Asp Leu 205 210 215 ACA CAG GTG AGC GAG GCA GCC GGG CCCCTT GTC CCA CCA CCC GCA CTT 2126 Thr Gln Val Ser Glu Ala Ala Gly Pro LeuVal Pro Pro Pro Ala Leu 220 225 230 235 CCC AAC TGC ACA GCC TCG AGT AGAACC CTG GAT CCG GCT TGT TAA 2171 Pro Asn Cys Thr Ala Ser Ser Arg Thr LeuAsp Pro Ala Cys * 240 245 250 AGGACCAAAG GGCATCTAAC AGCTTCTAG 2200 123amino acids amino acid linear protein 10 Met Asn Gly Ser Gly Ser Gln GlyAla Glu Asn Thr Ser Gln Glu Gly 1 5 10 15 Gly Ser Gly Gly Trp Gln ProGlu Ala Val Leu Val Pro Leu Phe Phe 20 25 30 Ala Leu Ile Phe Leu Val GlyThr Val Gly Asn Ala Leu Val Leu Ala 35 40 45 Val Leu Leu Arg Gly Gly GlnAla Val Ser Thr Thr Asn Leu Phe Ile 50 55 60 Leu Asn Leu Gly Val Ala AspLeu Cys Phe Ile Leu Cys Cys Val Pro 65 70 75 80 Phe Gln Ala Thr Ile TyrThr Leu Asp Asp Trp Val Phe Gly Ser Leu 85 90 95 Leu Cys Lys Ala Val HisPhe Leu Ile Phe Leu Thr Met His Ala Ser 100 105 110 Ser Phe Thr Leu AlaAla Val Ser Leu Asp Arg 115 120 249 amino acids amino acid linearprotein 11 Tyr Leu Ala Ile Arg Tyr Pro Leu His Ser Arg Glu Leu Arg ThrPro 1 5 10 15 Arg Asn Ala Leu Ala Ala Ile Gly Leu Ile Trp Gly Leu AlaLeu Leu 20 25 30 Phe Ser Gly Pro Tyr Leu Ser Tyr Tyr Arg Gln Ser Gln LeuAla Asn 35 40 45 Leu Thr Val Cys His Pro Ala Trp Ser Ala Pro Arg Arg ArgAla Met 50 55 60 Asp Leu Cys Thr Phe Val Phe Ser Tyr Leu Leu Pro Val LeuVal Leu 65 70 75 80 Ser Leu Thr Tyr Ala Arg Thr Leu Arg Tyr Leu Trp ArgThr Val Asp 85 90 95 Pro Val Thr Ala Gly Ser Gly Ser Gln Arg Ala Lys ArgLys Val Thr 100 105 110 Arg Met Ile Ile Ile Val Ala Val Leu Phe Cys LeuCys Trp Met Pro 115 120 125 His His Ala Leu Ile Leu Cys Val Trp Phe GlyArg Phe Pro Leu Thr 130 135 140 Arg Ala Thr Tyr Ala Leu Arg Ile Leu SerHis Leu Val Ser Tyr Ala 145 150 155 160 Asn Ser Cys Val Asn Pro Ile ValTyr Ala Leu Val Ser Lys His Phe 165 170 175 Arg Lys Gly Phe Arg Lys IleCys Ala Gly Leu Leu Arg Pro Ala Pro 180 185 190 Arg Arg Ala Ser Gly ArgVal Ser Ile Leu Ala Pro Gly Asn His Ser 195 200 205 Gly Ser Met Leu GluGln Glu Ser Thr Asp Leu Thr Gln Val Ser Glu 210 215 220 Ala Ala Gly ProLeu Val Pro Pro Pro Ala Leu Pro Asn Cys Thr Ala 225 230 235 240 Ser SerArg Thr Leu Asp Pro Ala Cys 245 250 26 base pairs nucleic acid singlelinear DNA 12 CAAGGCTGTT CATTTCCTCA TCTTTC 26 25 base pairs nucleic acidsingle linear DNA 13 TTGGAGACCA GAGCGTAAAC GATGG 25 45 base pairsnucleic acid single linear DNA 14 AGTCGACCCG GTGACTGCAG GCTCAGGTTCCCAGCGCGCC AAACG 45 23 base pairs nucleic acid single linear DNA 15CCCTACCTGA GCTACTACCG TCA 23 24 base pairs nucleic acid single linearDNA 16 ACCAAACCAC ACGCAGAGGA TAAG 24 26 base pairs nucleic acid singlelinear DNA 17 CCACGATGAG GATCATGCGT GTCACC 26 26 base pairs nucleic acidsingle linear DNA 18 TAGGTCAGGC CGAGAACCAG CACAGG 26 24 base pairsnucleic acid single linear DNA 19 CAGGTAGCTG AAGACGAAGG TGCA 24 25 basepairs nucleic acid single linear DNA 20 CTGCACCTTC GTCTTCAGCT ACCTG 2526 base pairs nucleic acid single linear DNA 21 CCTGTGCTGG TTCTCGGCCTGACCTA 26 26 base pairs nucleic acid single linear DNA 22 TATCTGGCCATCCGCTACCC GCTGCA 26 45 base pairs nucleic acid single linear DNA 23TTGCGCTACC TCTGGCGCGC CGTCGACCCG GTGGCCGCGG GCTCG 45 25 base pairsnucleic acid single linear DNA 24 CCAACAATGA CTCCAACTCT GTGAC 25 25 basepairs nucleic acid single linear DNA 25 AGGCGCAGAA CTGGTAGGTA TGGAA 2525 base pairs nucleic acid single linear DNA 26 AAGCTTCTAG AGATCCCTCGACCTC 25 28 base pairs nucleic acid single linear DNA 27 ACGGAATTCGACATGAATGG CTCCGGCA 28 34 base pairs nucleic acid single linear DNA 28GCTCTAGAGC CCCTTTGGTC CTTTAACAAG CCGG 34 1365 base pairs nucleic acidsingle linear DNA CDS 102..1265 29 AGTCGCACTA GGAGTTGCAG CGGCCGCAGCCCCGGGAGCT TCCCGCTCGC GGAGACCCAG 60 ACGGCTGCAG GAGCCCGGGC AGCCTCGGGGTCAGCGGCAC C ATG AAC GTC TCG 113 Met Asn Val Ser 1 GGC TGC CCA GGG GCCGGG AAC GCG AGC CAG GCG GGC GGC GGG GGA GGC 161 Gly Cys Pro Gly Ala GlyAsn Ala Ser Gln Ala Gly Gly Gly Gly Gly 5 10 15 20 TGG CAC CCC GAG GCGGTC ATC GTG CCC CTG CTC TTC GCG CTC ATC TTC 209 Trp His Pro Glu Ala ValIle Val Pro Leu Leu Phe Ala Leu Ile Phe 25 30 35 CTC GTG GGC ACC GTG GGCAAC ACG CTG GTG CTG GCG GTG CTG CTG CGC 257 Leu Val Gly Thr Val Gly AsnThr Leu Val Leu Ala Val Leu Leu Arg 40 45 50 GGC GGC CAG GCG GTC AGC ACTACC AAC CTG TTC ATC CTT AAC CTG GGC 305 Gly Gly Gln Ala Val Ser Thr ThrAsn Leu Phe Ile Leu Asn Leu Gly 55 60 65 GTG GCC GAC CTG TGT TTC ATC CTGTGC TGC GTG CCC TTC CAG GCC ACC 353 Val Ala Asp Leu Cys Phe Ile Leu CysCys Val Pro Phe Gln Ala Thr 70 75 80 ATC TAC ACC CTG GAC GGC TGG GTG TTCGGC TCG CTG CTG TGC AAG GCG 401 Ile Tyr Thr Leu Asp Gly Trp Val Phe GlySer Leu Leu Cys Lys Ala 85 90 95 100 GTG CAC TTC CTC ATC TTC CTC ACC ATGCAC GCC AGC AGC TTC ACG CTG 449 Val His Phe Leu Ile Phe Leu Thr Met HisAla Ser Ser Phe Thr Leu 105 110 115 GCC GCC GTC TCC CTG GAC AGG TAT CTGGCC ATC CGC TAC CCG CTG CAC 497 Ala Ala Val Ser Leu Asp Arg Tyr Leu AlaIle Arg Tyr Pro Leu His 120 125 130 TCC CGC GAG CTG CGC ACG CCT CGA AACGCG CTG GCA GCC ATC GGG CTC 545 Ser Arg Glu Leu Arg Thr Pro Arg Asn AlaLeu Ala Ala Ile Gly Leu 135 140 145 ATC TGG GGG CTG TCG CTG CTC TTC TCCGGG CCC TAC CTG AGC TAC TAC 593 Ile Trp Gly Leu Ser Leu Leu Phe Ser GlyPro Tyr Leu Ser Tyr Tyr 150 155 160 CGC CAG TCG CAG CTG GCC AAC CTG ACCGTG TGC CAT CCC GCG TGG AGC 641 Arg Gln Ser Gln Leu Ala Asn Leu Thr ValCys His Pro Ala Trp Ser 165 170 175 180 GCC CCT CGC CGC CGC GCC ATG GACATC TGC ACC TTC GTC TTC AGC TAC 689 Ala Pro Arg Arg Arg Ala Met Asp IleCys Thr Phe Val Phe Ser Tyr 185 190 195 CTG CTT CCT GTG CTG GTT CTC GGCCTG ACC TAC GCG CGC ACC TTG CGC 737 Leu Leu Pro Val Leu Val Leu Gly LeuThr Tyr Ala Arg Thr Leu Arg 200 205 210 TAC CTC TGG CGC GCC GTC GAC CCGGTG GCC GCG GGC TCG GGT GCC CGG 785 Tyr Leu Trp Arg Ala Val Asp Pro ValAla Ala Gly Ser Gly Ala Arg 215 220 225 CGC GCC AAG CGC AAG GTG ACA CGCATG ATC CTC ATC GTG GCC GCG CTC 833 Arg Ala Lys Arg Lys Val Thr Arg MetIle Leu Ile Val Ala Ala Leu 230 235 240 TTC TGC CTC TGC TGG ATG CCC CACCAC GCG CTC ATC CTC TGC GTG TGG 881 Phe Cys Leu Cys Trp Met Pro His HisAla Leu Ile Leu Cys Val Trp 245 250 255 260 TTC GGC CAG TTC CCG CTC ACGCGC GCC ACT TAT GCG CTT CGC ATC CTC 929 Phe Gly Gln Phe Pro Leu Thr ArgAla Thr Tyr Ala Leu Arg Ile Leu 265 270 275 TCG CAC CTG GTC TCC TAC GCCAAC TCC TGC GTC AAC CCC ATC GTT TAC 977 Ser His Leu Val Ser Tyr Ala AsnSer Cys Val Asn Pro Ile Val Tyr 280 285 290 GCG CTG GTC TCC AAG CAC TTCCGC AAA GGC TTC CGC ACG ATC TGC GCG 1025 Ala Leu Val Ser Lys His Phe ArgLys Gly Phe Arg Thr Ile Cys Ala 295 300 305 GGC CTG CTG GGC CGT GCC CCAGGC CGA GCC TCG GGC CGT GTG TGC GCT 1073 Gly Leu Leu Gly Arg Ala Pro GlyArg Ala Ser Gly Arg Val Cys Ala 310 315 320 GCC GCG CGG GGC ACC CAC AGTGGC AGC GTG TTG GAG CGC GAG TCC AGC 1121 Ala Ala Arg Gly Thr His Ser GlySer Val Leu Glu Arg Glu Ser Ser 325 330 335 340 GAC CTG TTG CAC ATG AGCGAG GCG GCG GGG GCC CTT CGT CCC TGC CCC 1169 Asp Leu Leu His Met Ser GluAla Ala Gly Ala Leu Arg Pro Cys Pro 345 350 355 GGC GCT TCC CAG CCA TGCATC CTC GAG CCC TGT CCT GGC CCG TCC TGG 1217 Gly Ala Ser Gln Pro Cys IleLeu Glu Pro Cys Pro Gly Pro Ser Trp 360 365 370 CAG GGC CCA AAG GCA GGCGAC AGC ATC CTG ACG GTT GAT GTG GCC TGA 1265 Gln Gly Pro Lys Ala Gly AspSer Ile Leu Thr Val Asp Val Ala * 375 380 385 AAGCACTTAGCGGGCGCGCTGGGATGTCACAGAGTTGGAGTCATTGTTGGGGGACCGTGG 1325GGAGAGCTTTGCCTGTTAATAAAACGCACAAACCATTTCA 1365 387 amino acids amino acidlinear protein 30 Met Asn Val Ser Gly Cys Pro Gly Ala Gly Asn Ala SerGln Ala Gly 1 5 10 15 Gly Gly Gly Gly Trp His Pro Glu Ala Val Ile ValPro Leu Leu Phe 20 25 30 Ala Leu Ile Phe Leu Val Gly Thr Val Gly Asn ThrLeu Val Leu Ala 35 40 45 Val Leu Leu Arg Gly Gly Gln Ala Val Ser Thr ThrAsn Leu Phe Ile 50 55 60 Leu Asn Leu Gly Val Ala Asp Leu Cys Phe Ile LeuCys Cys Val Pro 65 70 75 80 Phe Gln Ala Thr Ile Tyr Thr Leu Asp Gly TrpVal Phe Gly Ser Leu 85 90 95 Leu Cys Lys Ala Val His Phe Leu Ile Phe LeuThr Met His Ala Ser 100 105 110 Ser Phe Thr Leu Ala Ala Val Ser Leu AspArg Tyr Leu Ala Ile Arg 115 120 125 Tyr Pro Leu His Ser Arg Glu Leu ArgThr Pro Arg Asn Ala Leu Ala 130 135 140 Ala Ile Gly Leu Ile Trp Gly LeuSer Leu Leu Phe Ser Gly Pro Tyr 145 150 155 160 Leu Ser Tyr Tyr Arg GlnSer Gln Leu Ala Asn Leu Thr Val Cys His 165 170 175 Pro Ala Trp Ser AlaPro Arg Arg Arg Ala Met Asp Ile Cys Thr Phe 180 185 190 Val Phe Ser TyrLeu Leu Pro Val Leu Val Leu Gly Leu Thr Tyr Ala 195 200 205 Arg Thr LeuArg Tyr Leu Trp Arg Ala Val Asp Pro Val Ala Ala Gly 210 215 220 Ser GlyAla Arg Arg Ala Lys Arg Lys Val Thr Arg Met Ile Leu Ile 225 230 235 240Val Ala Ala Leu Phe Cys Leu Cys Trp Met Pro His His Ala Leu Ile 245 250255 Leu Cys Val Trp Phe Gly Gln Phe Pro Leu Thr Arg Ala Thr Tyr Ala 260265 270 Leu Arg Ile Leu Ser His Leu Val Ser Tyr Ala Asn Ser Cys Val Asn275 280 285 Pro Ile Val Tyr Ala Leu Val Ser Lys His Phe Arg Lys Gly PheArg 290 295 300 Thr Ile Cys Ala Gly Leu Leu Gly Arg Ala Pro Gly Arg AlaSer Gly 305 310 315 320 Arg Val Cys Ala Ala Ala Arg Gly Thr His Ser GlySer Val Leu Glu 325 330 335 Arg Glu Ser Ser Asp Leu Leu His Met Ser GluAla Ala Gly Ala Leu 340 345 350 Arg Pro Cys Pro Gly Ala Ser Gln Pro CysIle Leu Glu Pro Cys Pro 355 360 365 Gly Pro Ser Trp Gln Gly Pro Lys AlaGly Asp Ser Ile Leu Thr Val 370 375 380 Asp Val Ala 385 1384 base pairsnucleic acid single linear DNA 31GTGAGCCAGCGCCTTGGCCTCCCTGGGAGATGGGCATCCACGCGGGGGATGGAGCGGGAG 60GCGGGACTGGGGACCAAGAAGGGACGCGCAGAGTGGGACAGGACACTAAG AAGGCAGTGG 120AAGACAAGCGGGCGCGGAGGAGGAAAAAGAGGAATAAGAATGGGGGACCG TGGTGTCCCT 180CGGTTAGATGCGTCCTGGGGCCTGGAAGCCTGGAGAATGTGGCTCTCCAGCGCCGCCCGT 240GCCTGACAACGCGCAGCGTTTCCCAGTACGACGCGTTTGTGCGCGTTCATCTCGCTTGAG 300CTTAATGCCCTCCGTGAGGGTGGGATAGGACAAAGTGCCCAATATACAGA AGAGTTGAGT 360TCCTAAGTAACTCGCTCAGAGTCGCCAGCCAGGGATCGGGTGCGTGAAGTGACCGTCTGT 420CTCCTGCAGCCAACTTCAGGCGCCTCCACTGCGCTCGCCTCCAAGCCACGGTTTGGTTGG 480TTGGTGCAGCTGGCTCAGGTCCAGGCTGTGGATCTTGGGTCCTTTGCAAGGATCCACTCC 540GGAGTCCCAGCGAGCGTGCCTAAAGGTCCCTAGCTCAGTCCCAGCCCACTCTGCCTCTCG 600CCTCCAAACAAAACAAAACAAAATAAAATCCAAAACAAGTCGGGGCCGGGAGAGGAGCGT 660GCCCTGGGGTTCTTCCTCCCCAGCCAGAGGAGAGCGAGAGACGCACATTCGGGAGAGCGC 720GGGACTCAGGTGGAGCTTGAAAGGACACTGGGATGGTTCCTGGGGAGGAAATCCGGGTAT 780TTCCCCTCTCCATCCTCTGGAAAAACAGAGAGGCGAGGCCAGACTGCCCCCACACCTCCT 840GTAGCCACTGAGCGCGAAGTGCGTTGGTTCCGAGCGCGCTGGTGGGATCCACAAAGCTCG 900CATTCTCTCAGGAATCCCCTGAGAAATTAACTGTCCCTTGCCCAACATGTCTTCTCCAGG 960CTGTCTGCTAGAGCCTCAGGCGCCTCCGCCCTCCCTCCCGCGGCACCGTCACCAGTGGGT 1020AGTCACAGCCTCCCGGAGCCCATAGCCGGTTCTCCAACCTTTAGTCTTCAGTGGCTTTGG 1080GGTGCCCTCTCAGTGGAGACTGTGGTTGCAGTCCCCGGGGGCAGCGGGAGAATGGCTTGA 1140AGGCACACCTTTCCTGCTGCCGGGCCCGCCCCATTTCCAGGCTCCGCTGAGTGTCTGGGA 1200CACGCTGGGAGGCCCCCACCTCCGCCCTCACGCCGAGCCTCACCCCCACCTCCTCTGTGT 1260GCGGTGTAACCATGCGCTAAGGACCTTCCTCGAGAGCAGCCTTGGGACCGAGGTGCAGGG 1320GTCGCGGCCCTCCAGCATGAATGTGCCCGCTCAGCCGACGTCTCCCTTCCCGGTCTGACC 1380 GCAG1384 346 amino acids amino acid single linear DNA 32 Met Glu Leu Ala ProVal Asn Leu Ser Glu Gly Asn Gly Ser Asp Pro 1 5 10 15 Glu Pro Pro AlaGlu Pro Arg Pro Leu Phe Gly Ile Gly Val Glu Asn 20 25 30 Phe Ile Thr LeuVal Val Phe Gly Leu Ile Phe Ala Met Gly Val Leu 35 40 45 Gly Asn Ser LeuVal Ile Thr Val Leu Ala Arg Ser Lys Pro Gly Lys 50 55 60 Pro Arg Ser ThrThr Asn Leu Phe Ile Leu Asn Leu Ser Ile Ala Asp 65 70 75 80 Leu Ala TyrLeu Leu Phe Cys Ile Pro Phe Gln Ala Thr Val Tyr Ala 85 90 95 Leu Pro ThrTrp Val Leu Gly Ala Phe Ile Cys Lys Phe Ile His Tyr 100 105 110 Phe PheThr Val Ser Met Leu Val Ser Ile Phe Thr Leu Ala Ala Met 115 120 125 SerVal Asp Arg Tyr Val Ala Ile Val His Ser Arg Arg Ser Ser Ser 130 135 140Leu Arg Val Ser Arg Asn Ala Leu Leu Gly Val Gly Phe Ile Trp Ala 145 150155 160 Leu Ser Ile Ala Met Ala Ser Pro Val Ala Tyr Tyr Gln Arg Leu Phe165 170 175 His Arg Asp Ser Asn Gln Thr Phe Cys Trp Glu His Trp Pro AsnGln 180 185 190 Leu His Lys Lys Ala Tyr Val Val Cys Thr Phe Val Phe GlyTyr Leu 195 200 205 Leu Pro Leu Leu Leu Ile Cys Phe Cys Tyr Ala Lys ValLeu Asn His 210 215 220 Leu His Lys Lys Leu Lys Asn Met Ser Lys Lys SerGlu Ala Ser Lys 225 230 235 240 Lys Lys Thr Ala Gln Thr Val Leu Val ValVal Val Val Phe Gly Ile 245 250 255 Ser Trp Leu Pro His His Val Ile HisLeu Trp Ala Glu Phe Gly Ala 260 265 270 Phe Pro Leu Thr Pro Ala Ser PhePhe Phe Arg Ile Thr Ala His Cys 275 280 285 Leu Ala Tyr Ser Asn Ser SerVal Asn Pro Ile Ile Tyr Ala Phe Leu 290 295 300 Ser Glu Asn Phe Arg LysAla Tyr Lys Gln Val Phe Lys Cys Arg Val 305 310 315 320 Cys Asn Glu SerPro His Gly Asp Ala Lys Glu Lys Asn Arg Ile Asp 325 330 335 Thr Pro ProSer Thr Asn Cys Thr His Val 340 345

What is claimed is:
 1. A process for identifying a chemical compoundwhich specifically binds to a human galanin receptor (GALR2) whichcomprises contacting cells transfected with DNA encoding and expressingon their cell surface the human galanin receptor (GALR2), wherein suchcells prior to being transfected with such DNA do not express the humangalanin receptor (GALR2), with the compound under conditions suitablefor binding, and detecting specific binding of the chemical compound tothe human galanin receptor (GALR2), wherein the human galanin receptor(GALR2) has an amino acid sequence identical to that (a) shown in SEQ IDNO: 30, (b) expressed by COS-7 cells transfected with plasmid BO29 (ATCCAccession No. 97735) or (c) expressed by COS-7 cells transfected withplasmid BO39 (ATCC Accession No. 97851).
 2. A process for identifying achemical compound which specifically binds to a human or rat galaninreceptor (GALR2) which comprises contacting a membrane fraction fromcells transfected with DNA encoding and expressing on their cell surfacethe human galanin receptor (GALR2) or the rat galanin receptor (GALR2),wherein such cells prior to being transfected with such DNA do notexpress the human or rat galanin receptor (GALR2), with the compoundunder conditions suitable for binding, and detecting specific binding ofthe chemical compound to the human or rat galanin receptor (GALR2),wherein the human galanin receptor (GALR2) has an amino acid sequenceidentical to that (a) shown in SEQ ID NO: 30, (b) expressed by COS-7cells transfected with plasmid BO29 (ATCC Accession No. 97735) orexpressed by COS-7 cells transfected with plasmid BO39 (ATCC AccessionNo. 97851), and the rat galanin receptor (GALR2) has an amino acidsequence identical to that (a) shown in SEQ ID NO: 8, (b) expressed byLM(tk−) cells transfected with plasmid K985 (ATCC Accession No. 97426)or (c) expressed by LM(tk−) cells transfected with plasmid K1045 (ATCCAccession No. 97778).
 3. The process of claim 1 or 2, wherein the cellsare insect cells.
 4. The process of claim 1 or 2, wherein the cells aremammalian cells.
 5. The process of claim 4, wherein the cells arenonneuronal in origin.
 6. The process of claim 5, wherein chenonneuronal cells are COS-7 cells, 293 human embryonic kidney cells, CHOcells, NIH-3T3 cells or LM(tk−) cells.
 7. The process of claim 5,wherein the nonneuronal cells are LM(tk−) cells designated L-rGALR2-8(ATCC Accession No. CRL-12074).
 8. The process of claim 5, wherein thenonneuronal cells are LM(tk−) cells designated L-rGALR2I-4 (ATCCAccession No. CRL-12223).
 9. The process of claim 5, wherein thenonneuronal cells are CHO cells designated C-rGalR2-79 (ATCC AccessionNo. CRL-12262).
 10. The process of claim 4, wherein the compound is notpreviously known to bind to a human or a rat galanin receptor (GALRA2).11. A process involving competitive binding for identifying a chemicalcompound which specifically binds to a human galanin receptor (GALR2)which comprises separately contacting cells transfected with DNAencoding and expressing on their cell surface the human galanin receptor(GALR2), wherein such cells prior to being transfected with such DNA donot express the human galanin receptor (GALR2), with both the chemicalcompound and a second chemical compound known to bind to the receptor,and with only the second chemical compound, under conditions suitablefor binding of both compounds, and detecting specific binding of thechemical compound to the human galanin receptor (GALR2), a decrease inthe binding of the second chemical compound to the human galaninreceptor (GALR2) in the presence of the chemical compound indicatingthat the chemical compound binds to the human galanin receptor (GALR2),wherein the human galanin receptor (GALR2) has an amino acid sequenceidentical to that (a) shown in SEQ ID NO: 30, (b) expressed by COS-7cells transfected with plasmid BO29 (ATCC Accession No. 97735) or (c)expressed by COS-7 cells transfected with plasmid BO39 (ATCC AccessionNo. 97851).
 12. A process involving competitive binding for identifyinga chemical compound which specifically binds to a human or rat galaninreceptor (GALR2) which comprises separately contacting a membranefraction from a cell extract of cells transfected with DNA encoding andexpressing on their cell surface the human or rat galanin receptor(GALR2), wherein such cells prior to being transfected with such DNA donot express the human or rat galanin receptor (GALR2), with both thechemical compound and a second chemical compound known to bind to thereceptor, and with only the second chemical compound, under conditionssuitable for binding of both compounds, and detecting specific bindingof the chemical compound to the human or rat galanin receptor (GALR2), adecrease in the binding of the second chemical compound to the human orrat galanin receptor (GALR2) in the presence of the chemical compoundindicating that the chemical compound binds to the human or rat galaninreceptor (GALR2), wherein the human galanin receptor (GALR2) has anamino acid sequence identical to that (a) shown in SEQ ID NO: 30, (b)expressed by COS-7 cells transfected with plasmid BO29 (ATCC AccessionNo. 97735) or (c) expressed by COS-7 cells transfected with plasmid BO39(ATCC Accession No. 97851), and the rat galanin receptor (GALR2) has anamino acid sequence identical to that (a) shown in SEQ ID NO: 8, (b)expressed by LM(tk−) cells transfected with plasmid K985 (ATCC AccessionNo. 97426) or (c) expressed by LM(tk−) cells transfected with plasmidK1045 (ATCC Accession No. 97778).
 13. A method of screening a pluralityof chemical compounds not known to bind to a human or rat galaninreceptor (GALR2) to identify a compound which specifically binds to thehuman or rat galanin receptor (GALR2), which comprises (a) contactingcells transfected with and expressing DNA encoding the human or ratgalanin receptor (GALR2), wherein such cells prior to being transfectedwith such DNA do not express the human or rat galanin receptor (GALR2),with a compound known to bind specifically to the human or rat galaninreceptor (GALR2); (b) contacting the preparation of step (a) with theplurality of compounds not known to bind specifically to the human orrat galanin receptor (GALR2), under conditions permitting binding ofcompounds known to bind the human or rat galanin receptor (GALR2); (c)determining whether the binding of the compound known to bind to thehuman or rat galanin receptor (GALR2) is reduced in the presence of thecompounds, relative to the binding of the compound in the absence of theplurality of compounds; and if so (d) separately determining the bindingto the human or rat galanin receptor (GALR2) of each compound includedin the plurality of compounds, so as to thereby identify the compoundwhich specifically binds to the human or rat galanin receptor (GALR2);wherein the human galanin receptor (GALR2) has an amino acid sequenceidentical to that (a) shown in SEQ ID NO: 30, (b) expressed by COS-7cells transfected with plasmid BO29 (ATCC Accession No. 97735) or (c)expressed by COS-7 cells transfected with plasmid BO39 (ATCC AccessionNo. 37851), and the rat galanin receptor (GALR2) has an amino acidsequence identical to that (a) shown in SEQ ID NO: 8, (b) expressed byLM(tk−) cells transfected with plasmid K985 (ATCC Accession No. 97426)or (c) expressed by LM(tk−) cells transfected with plasmid K1045 (ATCCAccession No. 9778).
 14. A method of screening a plurality of chemicalcompounds not known to bind to a human or rat galanin receptor (GALR2)to identify a compound which specifically binds to the human or ratgalanin receptor (GALR2), which comprises (a) preparing a cell extractfrom cells transfected with and expressing DNA encoding the human or ratgalanin receptor (GALR2), wherein such cells prior to being transfectedwith such DNA do not express the human or rat galanin receptor (GALR2),isolating a membrane fraction from the cell extract, contacting themembrane fraction with a compound known to bind specifically to thehuman or rat galanin receptor (GALR2); (b) contacting the preparation ofstep (a) with the plurality of compounds not known to bind specificallyto the human or rat galanin receptor (GALR2), under conditionspermitting binding of compounds known to bind the human or rat galaninreceptor (GALR2); (c) determining whether the binding of the compoundknown to bind to the human or rat galanin receptor (GALR2) is reduced inthe presence of the compounds, relative to the binding of the compoundin the absence of the plurality of compounds; and if so (d) separatelydetermining the binding to the human or rat galanin receptor (GALR2) ofeach compound included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the human or ratgalanin receptor (GALR2); wherein the human galanin receptor (GALR2) hasan amino acid sequence identical to that (a) shown in SEQ ID NO: 30, (b)expressed by COS-7 cells transfected with plasmid BO29 (ATCC AccessionNo. 97735) or (c) expressed by COS-7 cells transfected with plasmid BO39(ATCC Accession No. 97851), and the rat galanin receptor (GALR2) has anamino acid sequence identical to that (a) shown in SEQ ID NO: 8, (b)expressed by LM(tk−) cells transfected with plasmid K985 (ATCC AccessionNo. 97426) or (c) expressed by LM(tk−) cells transfected with plasmidK1045 (ATCC Accession No. 97778).
 15. The method of claim 13 or 14,wherein the cells are mammalian cells.
 16. The method of claim 15,wherein the mammalian cells are non-neuronal in origin.
 17. The methodof claim 16, wherein the non-neuronal cells are COS-7 cells, 293 humanembryonic kidney cells, CHO cells, LM(tk−) cells or NIH-3T3 cells. 18.The process of claim 5, wherein the nonneuronal cells are CHC cellsdesignated CHO-hGALR2-264 (ATCC Accession No. CRL-12379).
 19. Theprocess of claim 1 or 2, wherein the compound is not previously known tobind to a human or rat galanin receptor (GALR2).